Modified gangliosides and the functional derivatives thereof

ABSTRACT

N-acyl-N,N&#39;-di-lysogangliosides, N&#39;-acyl-N,N&#39;-di-lysogangliosides and N,N&#39;-diacyl-N,N&#39;-di-lysogangliosides, in which the acyl groups are derived from an organic acid of the aliphatic, aromatic, araliphatic, alicyclic or heterocyclic series and in which at least one of the two acyl groups is not aliphatic, and their preparation are disclosed. Also disclosed is the preparation of the esters, inner esters, amides and hydroxy peracylates of these compounds and salts thereof. These compounds are useful in the treatment of pathologies of the central and peripheral nervous systems.

This application is a divisional of application Ser. No. 07/611,700filed on Nov. 13, 1990, U.S. Pat. No. 5,264,424, the entire contents ofwhich are hereby incorporated by reference.

SUMMARY

The present invention concerns modified gangliosides and theirfunctional derivatives, and more preciselyN-acyl-N,N'-di-lysogangliosides, N'-acyl-N,N'-di-lysogangliosides andN,N'-diacyl-N,N'-di-lysogangliosides, in which the acyl groups arederived from an organic acid of the aliphatic, aromatic, araliphatic,alicyclic or heterocyclic series and in which at least one of the twoacyl groups is not aliphatic, their esters, inner esters, amides andhydroxy peracylates and their salts.

The invention is also directed to pharmaceutical preparations containingone or more of the aforesaid ganglioside derivatives or their salts, aswell as the therapeutic use thereof and methods for their preparation.

The basic ganglioside of the novel derivatives of the invention can beany one of those extracted from natural products and in particular fromthe central and peripheral nervous systems of vertebrates, but alsothose from the adrenal medulla, from erythrocytes, from the spleen orfrom other organs. They are preferably purified gangliosides, which,although not unitary chemical compounds, are identifiable by anapproximate formula including an oligosaccharide part, generallychemically well-defined for each ganglioside, a sialic part (that is,constituted by one or more sialic acids) and a ceramide part, the lasttwo parts generally constituted by a mixture of various sialic acids andvarious N-acyl-sphingosines varying in the lengths of their aliphaticchains and with various acyls derived from higher fatty acids. Theapproximate formula of such a ganglioside can be represented as follows##STR1## where the sialic acids have the general formula ##STR2## inwhich one or more of the primary or secondary hydroxy groups can also beacylated and in which the acyl groups are derived from acetic orglycolic acid and the "ceramide" residue corresponds to one of theformulae: ##STR3## in which n is 6-18 and the acyl group is derived froma saturated or unsaturated fatty acid having from 16 to 22 carbon atomsor from a corresponding hydroxy acid. The novel derivatives of theinvention differ with respect to the nature of this acyl. The acylmoiety is unitary therein, while in gangliosides it is a mixture derivedfrom various aliphatic acids having from 16 to 22 carbon atoms. Anotherdifference is that the acyl belongs to a series of carboxy acids whichare not naturally occurring, as opposed to gangliosides, that is, toacids of the aromatic, alicyclic or heterocyclic series. They aretherefore semisynthetic ganglioside derivatives containing an unnaturalceramide.

The number of sialic acids present in gangliosides usually varies from 1to 5. The sialic residues are bound to the oligosaccharide by aketose-type bond formed by the hydroxyl in the 2-position with ahydroxyl of the oligosaccharide.

When several sialic acids are bound together, the union of theirmolecules is brought about by ketose bonds formed between the hydroxylgroups at the 2-and 8-position of two sialic acid molecules. The sialicacids of gangliosides, including those which are purified as describedpreviously, are mixtures of various chemically unitary acids, forexample N-acetylneuraminic acid and N-glycolylneuraminic acid,predominantly the former, and possibly of one or more of their O-acylderivatives, for example, 8-O-acyl derivatives.

The oligosaccharide is composed of a maximum of 5 monosaccharides ortheir derivatives with an acylamino group, especially hexoses and theirderivatives of the aforesaid type. At least one glucose or galactosemolecule is however present in the oligosaccharide. The most frequentresidue as an acylamino derivative of the aforesaid sugars isN-acetylgucosamine and N-acetylgalactosamine.

To better illustrate the structure of the gangliosides included informula (I), which is essentially that of the derivatives of theinvention, and in particular the character of the bonds between thesaccharide part, the sialic acids and the ceramide, presented herewithin its entirety is the formula of a "pure" ganglioside GM₁ containingone single sialic acid (represented by N-acetylneuraminic orN-glycolylneuraminic acid): ##STR4##

The formula is essentially the same for derivatives of the ganglioside"GM₁ " according to the present invention with the ceramide residuesubstituted by a corresponding "artificial" ceramide, the N-acyl groupof which is derived from one of the acids of the aromatic, alicyclic orheterocyclic series.

The term "lysoganglioside" is used in the literature to designatecompounds derived from natural gangliosides by elimination of the acylgroup present on the sphingosine nitrogen therein. It can be eliminatedenzymatically, for example, by exposing the gangliosides to the actionof the glycosphingolipid-ceramide-deacylase enzyme. This type ofhydrolysis leaves intact the acylamino and acylhydroxy groups in theresidues of neuraminic acid. To deacylate these groups also, thusobtaining a ganglioside derivative containing two free amino groups,both on the sphingosine nitrogen and on the neuraminic nitrogen,chemical hydrolysis must be used, for example, with dilute potassium.hydroxide. The ganglioside derivatives obtained by deacylation on theneuraminic nitrogen in the manner described herein are usually known inthe literature by the term "de-N-acetyl-gangliosides", the acyl group inthis position being the acetyl group. Designating the two nitrogen atomsof the sphingosine residue and in the neuraminic residue as N and N'respectively, the term "N'-lysoganglioside" can also be used for theaforesaid de-N-acetyl-gangliosides, and similarly, the term"lysogangliosides" can be used for derivatives with the free amino groupin the sphingosine residue, which should therefore be more preciselyidentified by the term "N-lysogangliosides". The term"N,N'-di-lysogangliosides" refers, on the other hand, to the compoundwith both free amino groups. This nomenclature will be used throughoutthe present application.

The aforesaid definition of the derivatives according to the inventionincludes the group of ganglioside derivatives which present an acetylgroup on the neuraminic nitrogen and an aromatic, araliphatic, alicyclicor heterocyclic acyl on the sphingosine nitrogen.

The invention also includes N-acyl-lysogangliosides of this kind derivedfrom the aforesaid lysogangliosides obtained enzymatically and whichtherefore contain in their sialic acids the acyl groups present innatural gangliosides, and mixtures of acylamino groups derived mostlyfrom acetic acid and to a lesser extent glycolic acid, and possibly acylgroups which esterify the hydroxy groups. The term "N-lysogangliosides"or "N-acyl-lysogangliosides" will therefore be used in the followingdescription of the invention both for these derivatives, which will bequalified as "natural" (for example, natural N-lyso GM₁), and for thosewhich possess a unitary acetyl group on the neuraminic nitrogen, whichwill be designated without this description or preferably as derivativesof N,N'-di-lysogangliosides, for example,N-benzoyl-N'-acetyl-N,N'-di-lyso GM₃.

The term "acyl-dilysogangliosides" will hereafter be used to signify allthe new compounds of the invention. As will be detailed hereafter, it ispossible to selectively deacylate a ganglioside on the neuraminicnitrogen and on the hydroxy groups alone, for example with a dilutealkaline hydroxide. By acylating the amino group of the neuraminicresidue in these compounds with a different acyl from the acetyl (andglycolyl), N,N'-diacyl-N,N'-di-lysogangliosides are obtained which alsoconserve a natural part of gangliosides, that is, the mixed acyl groupderived from higher aliphatic acids on the sphingosine nitrogen. Thesederivatives, which constitute a preferred group of the new compoundsaccording to the present invention, will be designated asN'-acyl-N'-lysogangliosides, for example, N'-benzoyl-N'-lyso GM₁. It iswell known that gangliosides play an important role in the nervoussystem and it has recently been demonstrated that they are useful intherapy for pathologies of the peripheral nervous system and inpathologies of the central nervous system [Acta Psychiat. Scand., 55,102, (1977); Eur. Medicophys.,13, 1, (1977); Ric. Sci. Educ. Perm.Suppl. 9, 115, (1978 ); Adv. Exp. Med. Biol. 71, 275, (1976);Electromyogr. Clin. Neurophysiol., 19, 353, (1979); Minerva Medica, 69,3277, (1978); Minerva Stomat., 27, 177, (1978); Med. del Lavoro, 68, 296(1977); Brain Res. 197, 236, (1980)]. The therapeutic action ofgangliosides seems to consist mainly in stimulation of sproutingphenomena in nerve cells and in activating the enzymes involved in theconduction of nervous stimuli, such as the (Na⁺,K⁺) ATPase enzyme [BrainRes., 197, 236 (1980), J. of Neurochem. 37, 350 (1981)]. Neuronalsprouting stimulated by gangliosides enhances the functional recovery ofimpaired or damaged nerve tissue.

Further studies have been carried out to find compounds which may provemore efficient than gangliosides in therapies for nervous systempathologies. These studies have led for example to the discovery thatganglioside inner esters, in which one or more hydroxyls of thesaccharide part are esterified with one or more carboxy groups of thesialic acids (intramolecular reaction) with the formation of the samenumber of lactone rings, are more active than gangliosides themselves inenhancing neuronal sprouting and in activating the membrane enzymesinvolved in the conduction of nerve stimuli, such as the enzyme(Na⁺,K⁺)ATPase (see for example U.S. Pat. Nos. 4,476,119, 4,593,091 and4,716,223).

"Outer" esters of gangliosides, that is, esters of the carboxy functionsof the sialic acids with various alcohols of the aliphatic, araliphatic,alicyclic or heterocyclic series, also show an improved activity onneuronal sprouting and conduction of nervous stimuli. The amides ofgangliosides also possess the same properties, as do the peracylatedderivatives of amides, esters and simple gangliosides. All of thesederivatives, which are described in U.S. Pat. No. 4,713,374, are also tobe considered as basic substances for the acyl-di-lysogangliosides ofthe present invention.

The new compounds of the present invention are semisynthetic gangliosideanalogues and differ from the prior art molecules due to the presence ofN-acyl groups, both on the sphingosine nitrogen, and on the neuraminicnitrogen. They are not "natural", and therefore have at least one acylgroup derived from acids of the aromatic, araliphatic, alicyclic orheterocyclic series. Furthermore, they differ from natural gangliosides(with the aforesaid exception of "natural" N-acyl-N-lysogangliosides andN'-acyl-N'-lysogangliosides) because of the fact that the acyl groupsare unitary and well-defined. Those derivatives which contain adifferent acyl group from acetyl on the neuraminic nitrogen, can be acylgroups of the type present in natural gangliosides, that is, mixtures ofhigher fatty acids, such as stearic or palmitic acid.

At the basis of the present invention is the discovery that the new"semisynthetic" gangliosides also possess essentially the samepharmacological actions as natural gangliosides and their esters,amides, inner esters and peracylated derivatives of all of thesecompounds, with a range of action that is modified with respect to manyparameters, such as the rate of "onset", duration and intensity of thesprouting action of neuronal cells, and which can be regulated accordingto the greater or lesser lipophilic or hydrophilic character of the acylcomponent, or the type and extent of side effects, which in some casescan prove to be negative or positive, according to the therapeuticproblem being tackled. An example is the inhibiting action on proteinkinase C, which can be an undesirable and negative effect in certainconditions of imbalance of the normal mechanisms of neurotransmissionfunctions. Activation is triggered by an increased concentration ofexcitatory amino acids such as glutamic and/or aspartic acid. Theseacids have, under the aforesaid abnormal conditions, a direct toxicaction on neuronal cells. One great advantage of the products of thepresent invention, which sets them apart from other protein kinase Cinhibitors, such as gangliosides themselves or sphingosine, consists intheir ability to prevent and combat the aforesaid neurotoxic action.

It is important to emphasize that the products of the present invention,unlike calcium antagonists and glutamate receptor antagonists (NMDA inparticular), act only in the presence of abnormal conditions, andtherefore limit localized neurotoxicity and maintain neuronalplasticity, thereby allowing a more ready recovery of the impairedphysiological functions. In many cases it is possible to use thederivatives of the invention to make use of the action of the acidsthemselves, corresponding to a given acyl group, avoiding the specificaction of the ganglioside part, which in such cases functions as avehicle. This is the case, for example, with the new type of gangliosideill which the N-acyl group is derived from an acid which is active onthe central or peripheral nervous system, such as lysergic acid and itsanalogues, or nicotinic and isonicotinic acids. These acids have acertain action in vitro, but hardly any or no action at all in vivo.When they are introduced into the molecule of a ganglioside according tothe present invention, the action appears to its full extent in vivo.

The ganglioside derivatives of the present invention can therefore beused instead of natural products or the aforesaid already knownsemi-synthetic derivatives. They are of great value in cases of patientswho do not respond satisfactorily to conventional products or in caseswhich present individual idiosyncrasies or allergies. Moreover, they canbe used as vehicles because of the specific pharmacological action ofthe acid corresponding to the N-acyl group.

The lysogangliosides which serve as the base for the preparation of thenew acyl-di-lysogangliosides according to the present invention areabove all those obtainable by deacylation of gangliosides found innatural products, and in particular in tissues of the central andperipheral nervous systems of vertebrates, and also in adrenal medulla,erythrocytes, the spleen or in other organs. They can be purifiedgangliosides, such as those which are defined by this term in theliterature and are represented by a unitary structure with regard to thesaccharide part, or they can be mixtures of gangliosides. Among the mostimportant gangliosides to be used as starting bases for the derivativesof the invention can be mentioned, for example, those in which theoligosaccharide is formed by a maximum of 4 hexose residues, and inwhich this saccharide part is chemically unitary. The hexoses arepreferably chosen from the group formed by N-acetylglucosamine andN-acetylgalactosamine (ganglioside group A). The gangliosides of thisgroup are, for example, those extracted from the brains of vertebrates,such as those described in the article "Gangliosides of the NervousSystem" in "Glycolipid Methodology", Lloyd A., Witting Ed., American OilChemists Society, Champaign, Ill. 187-214 (1976 ) (see especially Table1), for example the gangliosides G_(M4), G_(M3), G_(M2), G_(M1) -GlcNAC,G_(D2), G_(D1a) -GalNAC, G_(T1c), G_(Q), and G_(T1) and, in particular,those in which the oligosaccharide contains at least one glucose residueor galactose residue and either N-acetylglucosamine orN-acetylgalactosamine and preferably the following (ganglioside groupB): ##STR5## where Glc stands for glucose, GalNAC stands forN-acetylgalactosamine, Gal stands for galactose, and NANA stands forN-acetylneuraminic acid.

The present invention also includes mixtures of the newN-acyl-lysogangliosides and in particular those which are derived fromthe ganglioside mixtures present in extracts from various animaltissues, such as in "total" extracts, or in various fractions, forexample those described in the literature. Examples of such literatureinclude the articles mentioned previously or the articles "Extractionand analysis of materials containing lipid bound sialic acid" in theaforesaid journal, pages 159-186 (1976) and in "Gangliosides of theNervous System" same publication, pages 187-214, and in the Germanpatent No. 2549680. In these new mixtures the N-acyl part of theganglioside mixtures is substituted by one of the aforesaid acyl groups,and they can be obtained according to the procedure of the presentinvention as disclosed hereafter by deacylation of the gangliosidemixtures and subsequent reacylation, optionally after the reacylation ofother deacylated groups in the sialic part of the gangliosides. Amongthe most important ganglioside mixtures to be used as starting productsare ganglioside extracts obtained from the nervous system, in particularfrom the brain and containing the gangliosides GM₁, G_(D1a), G_(D1b) andG_(T1b), already mentioned.

As noted above, at the basis of the present invention is the discoverythat the new semisynthetic ganglioside analogues described herein andtheir aforesaid functional derivatives or their salts possessessentially the same pharmacological actions as natural gangliosides ortheir analogous functional derivatives, with a range of action whichdiffers with regard to many parameters.

These modified gangliosides also possess an inhibiting action on proteinkinase C activation.

The aforesaid pharmacological properties of the modified gangliosides ofthe invention can be illustrated by the following experiments.

In primary neuronal cell cultures, stimulation of excitatory amino acid(EAA) receptors enhances the increase in Ca⁺² influx and translocation,with the consequent activation of protein kinase C (PKC) from thecytosol to the membranes. The addition of glutamate or exposure toanoxic conditions of primary cultures of granular cells induces celldamage leading to neuronal death. Acute cerebral ischemia is followed byan alteration in glutamergic transmission which triggers a cascade ofevents leading, as occurs in vitro, to cell death.

It is known that pre-exposure of primary neuronal cultures totrisialosyl-N-tetraglycosylceramide (GT_(1b)) ormonosialosyl-N-tetraglycosylceramide (GM₁) inhibits PKC translocationand protects against glutamate-induced cell death.

Binding tests have shown that the action mechanism of gangliosides isnot linked with receptor antagonism.

Reported here are the experiments conducted with the gangliosidederivatives N'-3,4,5-trimethoxybenzoyl-N'-lyso GM₁ (Ligade 5),N-(2-furoyl)-N-lyso GM₁ (Ligade 34),N-(1-methyl-2-pyrrol-carbonyl)-N-lyso GM₁ (Ligade 38),N-(2-thiopheneacetyl)-N-lyso GM₁ (Ligade 45),N,N'-di-phenylacetyl-di-lyso GM₁ (Ligade 82),N,N'-di-(2-pyridylacetyl)-di-lyso GM₁ (Ligade 84), andN,N'-di-(5-methyl-2-thiophenecarboxyl)-di-lyso GM₁ (Ligade 85), whichare suitable for the assessment of the capacity to antagonize selectiveneuronal death induced by glutamate.

MATERIALS AND METHODS Cell cultures

Primary cultures of cerebellar granule cells from 8-day-old SpragueDawley rats, composed of >90% of granule cells, approximately 5% ofGABAergic neurons and <5% of glial cells, were employed. In theseexperiments, cells were used on the 12th day of culture.

Induction of neurotoxicity with glutamate

The glutamate (100 μm in Locke's solution without Mg⁺²) was added to thecells and left to stand for 15 minutes at room temperature (controls hadno glutamate); the cultures were washed 3 times with Locke's solution toremove the excess glutamate, then replated in the original culturemedium.

Solubilization, incubation of the compound and method of analysis

Ligade 5, 34, 38, 45, 82, 84 and 85 were dissolved inchloroform/methanol 2:1, dried in N₂, resuspended in Locke's solutionplus Mg⁺² at a final concentration of 5×10⁻⁶ M and added to the culturesat 37° C. 15 minutes before induction of neurotoxicity. The GM₁,similarly solubilized, and used at a final concentration of 1×10⁻⁴ M,was added to the cells 120 minutes before exposure to L-GLU.

Cell survival was assessed 24 hrs later by the colorimetric method (D.O.570-630) using MTT(3-4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazolium).

RESULTS

The experiments showed that Ligade 5, 34, 38, 45, 82, 84 and 85, at aconcentration of 5×10⁻⁶ M and GM₁ at a concentration of 1×10⁻⁴ M used ascontrol, proved effective in protecting against glutamate-inducedneurotoxicity (p<0.05) (Table 1).

It should be noted that the Ligade derivatives are efficacious at doses10 times less than those required by GM₁ and after far shorterpreincubation times.

DISCUSSION

The results obtained clearly indicate that the new gangliosidederivatives, named Ligade 5, 34, 38, 45, 82, 84 and 85, are able toantagonize glutamate-induced neurotoxicity in primary cultures ofcerebellar granule cells.

The effect of the new derivatives is particularly interesting since itis observed at concentrations over 10 times less than those of GM₁ atcorresponding levels of efficacy, and after shorter periods ofpreincubation.

With regard to this effect, the derivatives of the invention can berecommended in acute and chronic pathologies based on glutamergic-typedamage, such as cerebral ischemia, trauma, epilepsy, chorea, Parkinson'sdisease, aging and dementia as well as brain disorders, hypoglycemia andhypoxia. Some of the mechanisms at the basis of brain damage, especiallywith regard to neurotoxicity, are however common to damage to othersystems too, such as the neurocardiovascular system.

                  TABLE 1                                                         ______________________________________                                        Protective effect of Ligade 5, 34, 38, 45, 82, 84 and                         85 and GM.sub.1 in a model of neurotoxicity induced by                        exogenous glutamate in primary cultures of cerebellar                         granule cells.                                                                                   cell survival                                              compound           MTT (DO 570-630)                                           ______________________________________                                        control Locke's solution-Mg.sup.+2                                                               0.156 ± 0.021                                           L-Glutamate        0.103 ± 0.004                                           L-Glu + Ligade 5   0.147 ± 0.014                                           L-Glu + Ligade 34  0.126 ± 0.003                                           L-Glu + Ligade 38  0.131 ± 0.003                                           L-Glu + Ligade 45  0.138 ± 0.006                                           L-Glu + Ligade 82  0.165 ± 0.006                                           L-Glu + Ligade 84  0.151 ± 0.007                                           L-Glu + Ligade 85  0.126 ± 0.008                                           L-Glu + GM.sup.1   0.133 ± 0.018                                           ______________________________________                                    

The granule cells were used on the 12th day of culture and were exposed,at room temperature, to 100 μM L-glutamate (L-GLU) for 15 minutes. TheLigade derivatives, solubilized in Locke's solution at a finalconcentration of 5×10⁻⁶ M, were added to the cells 15 minutes beforeinduction of neurotoxicity, while the GM₁ 1×10⁻⁴ M was pre-incubated for120 minutes. p<0.05 for GM₁ and Ligade derivatives vs. L-GLU.

In view of the pharmacological properties described above, the aforesaidsemisynthetic ganglioside analogues can be used as drugs in thefollowing pathologies: cerebral ischemia, metabolic encephalopathiessuch as hypoglycemia and hypoxia, encephalopathies of toxic origin,trauma, epilepsy, neurodegenerative diseases such as Parkinson's diseaseand Huntington's chorea, and mental disorders.

Administration is usually by injection, intramuscular, subcutaneous,intravenous, transdermal or pulmonary administration, preferably insuitably buffered aqueous solutions. Safe storage of the pharmaceuticalcan be ensured by preparing it in the form of vials containing solutionsof the derivative, optionally together with other auxiliary ingredients,as will shown hereafter in the case of the pharmaceutical preparationsof the present invention. For the therapeutic, or possibly alsopreventive, application by the aforesaid parenteral route, the dosagevaries preferably from 0.05 mg to 5 mg of active substance per kg ofbody weight/day and especially between 0.05 mg and 2 mg per kg of bodyweight/day.

Although the new therapeutic applications according to the invention aregenerally suitable for use in all pathologies connected with nerveconduction impairments in the central and peripheral nervous systems,the following can be specifically mentioned: retrobulbar opticalneuritis, paralysis of the oculomotor nerves, trigeminal neuralgia,paralysis of the facial nerve and Bell's palsy, Garcin's syndrome,traumatic lesions of the peripheral nerves, diabetic and alcoholicpolyneuritis, obstetrical paralysis, paralytic sciatica, motor neurondisease, amyotrophic lateral sclerosis, myelopathic muscular atrophy,progressive bulbar paralysis, myasthenia gravis and Lambert Eaton'ssyndrome, muscular dystrophy, impairments in synaptic nerve transmissionin the CNS and PNS, and consciousness deficiencies such as confusion,concussion, thrombosis and embolism.

The invention also includes the functional derivatives of the sialiccarboxy groups of the new acyl-lysogangliosides, that is, esters andamides, and also inner esters with lactone bonds between the sialiccarboxy groups and the hydroxyls of the oligosaccharide, similar tothose of gangliosides as well as the derivatives peracylated on theganglioside hydroxyls, both of acyl-lysogangliosides, and of theiraforesaid functional derivatives, and the salts of all the newacyl-di-lyso-gangliosides and of their functional derivatives. Thesesialic functional derivatives can be obtained from the newacyl-di-lysogangliosides by the procedures described in the variousaforesaid patents for the corresponding ganglioside derivatives.

The invention includes in particular also mixtures of these derivatives,such as are obtained from mixtures of acyl-lysogangliosides according tothe invention, obtained in turn from the aforesaid ganglioside mixtures.

The ester groups of the new N-acyl lysoganglioside derivatives of theinvention are derived particularly from alcohols of the aliphatic seriesand especially from those with a maximum of 12 and especially 6 carbonatoms, or of the araliphatic series with preferably only one benzenering, optionally substituted by 1-3 lower alkyl groups (C₁₋₄), forexample methyl groups, and a maximum of 4 carbon atoms in the aliphaticchain, or by alcohols of the alicyclic or aliphatic alicyclic serieswith only one cycloaliphatic ring and a maximum of 14 carbon atoms, orof the heterocyclic series with a maximum of 12 and especially 6 carbonatoms and only one heterocyclic ring containing a heteroatom chosen fromthe group formed by N, O and S. The amide groups of the carboxyfunctions in the N-acyl lysoganglioside derivatives of the presentinvention are derived from ammonia or from amines of any class, withpreferably a maximum of 12 carbon atoms.

The aforesaid alcohols and amines can be unsubstituted or substituted,especially by functions chosen from the group formed by hydroxy, aminoor alkoxy groups with a maximum of 4 carbon atoms in the alkyl, carboxyor carbalkoxy moiety with a maximum of 4 carbon atoms in the alkylresidue, or the alkylamino or dialkylamino group with a maximum of 4carbon atoms in the alkyl thereof, and can be saturated or unsaturated,especially with only one double bond.

The alcohols which esterify the carboxy functions of the N-acyllysogangliosides according to the present invention can be monovalent orpolyvalent, in particular bivalent. Of the alcohols of the aliphaticseries, special mention should be made of lower alcohols with a maximumof 6 carbon atoms, such as methyl alcohol, ethyl alcohol, propyl alcoholand isopropyl alcohol, n-butyl alcohol, isobutyl alcohol, and tert-butylalcohol, and of the bivalent alcohols, ethylene glycol and propyleneglycol. Of the alcohols of the araliphatic series, should be mentionedin particular those with one single benzene ring, such as benzyl alcoholand phenethyl alcohol. Of the alcohols of the alicyclic series,preference should be given to those with only one cycloaliphatic ring,such as cyclohexanol, or terpene alcohols, such as menthanol orcarvomenthol, or one of the terpineols or piperitol.

Of the alcohols of the heterocyclic series, special mention should bemade of tetrahydrofuranol or tetrahydropyranol. To esterify the carboxygroups of the N-acyl-lysogangliosides it is possible to use alsoaliphatic alcohols substituted, for example, by amino functions, such asaminoalcohols, for example those with a maximum of 4 carbon atoms andespecially aminoalcohols with a dialkyl (C₁₋₄) -amino group such asdiethylaminoethanol.

The carboxylamide functions according to the present invention areeither derived from ammonia (and the amide in this case is theunsubstituted amide --CONH₂) or from primary or secondary amines,especially from those containing a maximum of 12 carbon atoms. Suchamines can be of an aromatic, heterocyclic or alicyclic nature, but arepreferably aliphatic. Preferred objects of the present invention are thecarboxylamide derivatives of aliphatic amines with a maximum of 12carbon atoms, the amines of which can be open-chained, straight-chainedor branched or they can be cyclic, such as the alkylamines derived fromalkyls having from 1 to 6 carbon atoms, such as methylamine, ethylamine,propylamine, hexylamine, dimethylamine, diethylamine, di-isopropylamine,dihexylamine, or the alkyleneamines derived from alkylene groups withstraight chains having from 3 to 6 carbon atoms or corresponding chainssubstituted by 1 to 3 methyl groups, such as pyrrolidine, piperidine andazepine. The alkyl or alkylene groups of these amines can also beinterrupted in the carbon atom chain or substituted by otherhetero-atoms, in particular by nitrogen atoms. The amides of theinvention are derived in this case from diamines, such asethylenediamine, trimethylenediamine or piperazine. If the alkyl oralkylene groups are interrupted or substituted by oxygen or sulphuratoms, the amides represent derivatives of aminoalcohols, such asaminoethanol or aminopropanol or are derived from morpholine orthiomorpholine.

Of special interest in terms of the present invention are the aforesaidesters and amides of N-acyl lysogangliosides derived from gangliosidesof groups A and B mentioned above, and of their mixtures.

The invention also includes the derivatives peracylated in the hydroxylsof the saccharide part, sialic acids and ceramide of the esters andamides described herein. In such derivatives the acyl groups can bederived from acids of the aliphatic, aromatic, araliphatic, alicyclic orheterocyclic series. They are derived preferably from acids of thealiphatic series with a maximum of 10 carbon atoms and especially 6carbon atoms, such as formic acid, acetic acid, propionic acid, thebutyric acids, valerianic acids, capronic acid or caprinic acid. Theycan also be derived from acids, for example, with the same number ofcarbon atoms but substituted, particularly by hydroxyacids, such aslactic acid, by aminoacids such as glycine or by dibasic acids, such assuccinic, malonic or maleic acid.

Of the aromatic acids should be mentioned those with only one benzenenucleus, particularly benzoic acid and its derivatives with methyl,hydroxy, amino or carboxy groups, such as p-aminobenzoic acid, salicylicacid or phthalic acid.

The invention also includes the peracylated derivatives of N-acyllysogangliosides and their mixtures described previously, with, however,free carboxy functions. Of particular importance to these derivativesare those acylated derivatives derived from the acids listed herein. Onegroup of new derivatives to be specially mentioned is the oneconstituted by gangliosides esterified or converted into amides orperacylated on the hydroxyl groups, the ester groups of which arederived from aliphatic alcohols with a maximum of 6 saturated carbonatoms, unsubstituted or substituted by hydroxy, alkoxy groups with amaximum of 4 carbon atoms, amino, alkylamino or dialkylamino groups witha maximum of 4 carbon atoms in the alkyl groups, carboxy groups,carbalkoxy groups with a maximum of 4 carbon atoms in the alkyl residue,and by the corresponding alcohols with one double bond at the most, byaraliphatic alcohols with only one benzene ring, unsubstituted orsubstituted by 1 to 3 methyl groups, by cycloaliphatic oraliphatic-cycloaliphatic alcohols with a cyclohexane ring, unsubstitutedor substituted by 1 to 3 methyl groups and a maximum of 4 carbon atomsin the aliphatic part, by tetrahydrofuranol or by tetrahydropyranol.

The amide groups therein may be derived from ammonia or fromalkylamines, dialkylamines or alkyleneamines with a maximum of 6 carbonatoms in the alkyl groups and by 4 to 8 carbon atoms in the alkylenegroups and in which the alkyl or alkylene groups can be interrupted inthe carbon atom chain by heteroatoms chosen from the group formed bynitrogen, oxygen and sulphur, the amino group being possibly --NH-- incases where a nitrogen atom substituted by an alkyl with a maximum of 4carbon atoms is present and/or they may be substituted by groups chosenfrom the group formed by amino, alkylamino or dialkylamino groups with amaximum of 4 carbon atoms in the alkyl groups, or by hydroxy or alkoxygroups with a maximum of 4 carbon atoms in the alkyl group, or byaraliphatic amines with only one benzene ring, optionally substituted bya maximum of 3 methyl groups and with a maximum of 4 carbon atoms in thealiphatic part, and in which the acyl groups which esterify thehydroxyls are derived from saturated or unsaturated aliphatic acids witha maximum of 6 carbon atoms, which can also be substituted by a functionchosen from the groups formed by hydroxy, amino and carboxy groups, andby their salts.

Of the functional derivatives of the new semi-synthetic gangliosideanalogues should be mentioned especially the sialic esters of theaforesaid new compounds and derived from methyl, ethyl, propyl,isopropyl, n-butyl, isobutyl, tert-butyl, benzyl, allyl,ethoxycarbonylmethyl, or cyclohexyl alcohol, the sialic amides derivedfrom methylamine, ethylamine, propylamine, dimethylamine, diethylamine,pyrrolidine, piperidine, piperazine, morpholine, or thiomorpholine, andtheir peracylates, perpropionylates, perbutyrylates, permaleylates,persuccinylates and the peracylated analogues of the aforesaid sialicesters and amides.

The N-acyl radicals derived from an acid of the aromatic, alicyclic orheterocyclic series can have the cyclic system directly bound to thecarbamide group --NH --CO-- of the neuraminic or sphingosine residues ofthe ganglioside derivative, or by an aliphatic, alkylene or alkylideneresidue. The aforesaid terms therefore embrace, in the range of thepresent invention, both the derivatives of the aromatic, alicyclic orheterocyclic acids as such (that is, bound directly to the carbamidegroup) and the derivatives of araliphatic, aliphatic, alicyclic andaliphatic heterocyclic acid. The rings of these hydrocarbyl residues canof course in turn be substituted by aliphatic hydrocarbyl groups. Theaforesaid aliphatic chains can also be interrupted by heteroatoms, forexample, those chosen from the group formed by N, S and O. The cyclicsystems can be mono- or polycyclic, preferably, in the second case,bicyclic. The acids can be mono or polybasic, and preferably, in thesecond case, dibasic.

The N-acyl radicals of the compounds of the invention possess preferablyfrom 6 to 24carbon atoms and can contain one or more cyclic systems,preferably however only one, optionally substituted in their turn byaliphatic hydrocarbyl groups, especially alkyls, preferably with amaximum of 6 carbon atoms. The hydrocarbyl residues of the N-acyl groupscan also be substituted, both in the aliphatic parts and in the rings,by functions Or modified functions, such as especially halogens, forexample chlorine, bromine and fluorine, free or esterified hydroxygroups, free or esterified amino groups, or acylates, or substituted byalkyl or alkylene groups, free or catalyzed oxo-groups, oximes orsubstituted oximes, hydrazones or substituted hydrazones, free oretherified mercapto groups, free or substituted sulfamide groups, freeor esterified sulfonic groups, sulfoxide groups or nitryl or nitrogroups. The esters of the hydroxy or amino groups can be derived fromacids of the aliphatic, araliphatic, alicyclic or heterocyclic series.Such ester groups are derived above all from therapeutically acceptableacids. The aliphatic acids are preferably lower acids with a maximum of8 carbon atoms, such as acetic, propionic, butyric, or valerianic acid,for example isovalerianic acid, or their substituted derivatives such ashydroxyacids, for example glycolic acid, or α- or β-hydroxybutyric acid,lactic acid or aminoacids, for example-natural aminoacids such asglycine, alanine, valine or henylglycine, or dibasic acids, such asmalonic acid, succinic acid, maleic acid or malic acid, which also maybe optionally substituted.

Those of the aromatic series are, for example, benzoic acid or itsderivatives substituted by 1 to 3 lower alkyl groups, hydroxy or loweralkoxy groups, or by halogens such as chlorine, bromine or fluorine. Ofthe araliphatic acids should be mentioned primarily those with only onebenzene ring, such as phenylacetic or phenylpropionic acid, optionallysubstituted as previously described. Alicyclic acids are preferablythose with rings of 5 or 6 carbon atoms, such as cyclohexanecarboxylacid and cyclohexanedicarboxyl acid. Acids of the heterocyclic seriesare those reported hereafter, but are preferably simple acids with onlyone heterocyclic group, such as derivatives of pyridine, for example,nicotinic acid and isonicotinic acid or pyrrolidine-carboxyl acid.

Suitable alcohols which can represent the etherifying component of thehydroxy or mercapto etherifying groups are all those previously listedwith regard to the esters of the sialic carboxy groups, being part ofthe acyl-lysogangliosides of the present invention in the form of theirfunctional derivatives. Preferable are lower aliphatic or araliphaticalcohols with a maximum of 4 carbon atoms in the aliphatic part. Thealkyl or aralkyl groups as substituents on the amino groups or which arepresent in substituted ketal, acetal or keto groups or in esterifiedcarboxy groups preferably have a maximum of 4 carbon atoms in thealiphatic part and a benzene group optionally substituted as describedpreviously. The same maximum number of carbon atoms is also present inall the aliphatic groups designated as "lower" in the aforesaiddefinitions. A lower alkylene group, which can substitute amino groups,thus forming saturated heterocyclic groups, is constituted above all bythose having 4 or 5 carbon atoms.

Aromatic acyl groups are derived primarily from acids with only onearomatic ring, such as benzoic acid and its derivatives substituted byone or more, particularly by 1 to 3 groups, chosen from the group formedby alkyl, hydroxy, oxo, amino, mercapto, carboxy and sulfonic groups,free or functionally modified, or halogens, for example as describedabove. Examples thereof include benzoic, salicylic, p-aminobenzoic, thethree isomers of toluic acid, phthalic, isophthalic or terephthalicacid, p-hydroxybenzoic, protocatechuic, anisic, vanillic, veratric,piperonylic, resorcylic, orsellinic, pyrogallic, p-sulfaminebenzoic,2,6-dimethoxybenzoic, 3,4,5-trimethoxybenzoic, 2-chlorobenzoic,3-chlorobenzoic, 4-chlorobenzoic, 4-acetamidebenzoic,N-acetylanthranylic, 3-amino-benzoic, 4-aminobenzoic,2-amino-4-chlorobenzoic, 4-amino-2-chlorobenzoic,3-amino-4-methoxybenzoic, 4-butoxybenzoic, 4-butylbenzoic,2-chloro-5-methylthiobenzoic, 4-chlorophenoxyacetic,4-chloro-3-sulfamoylbenzoic, 4-cyanbenzoic, 2,3-dichlorobenzoic,2,4-dichlorobenzoic, 2,5-dichlorobenzoic, 2,6-dichlorobenzoic,3,4-dichlorobenzoic, 3,5-dichlorobenzoic, 4-diethylaminobenzoic,3,4-difluorobenzoic, 4-ethoxybenzoic, 2-fluorobenzoic, 4-fluorobenzoic,4-fluorophenoxyacetic, 4-heptyl-benzoic, 2-(4-hydroxyphenoxy) propionic,4-methylthiobenzoic, phenoxyacetic, 2-sulfobenzoic, andα-trifluoro-o-toluic acid.

These acyl groups can however also derive from acids with severalbenzene rings, condensed or not condensed, or with benzene rings andother cyclic hydrocarbyl residues, such as alicyclic or heterocyclicresidues, such as for example, naphthoic, p-aminonaphthoic,p-hydroxynaphthoic, naphthalic, diphenyl-o,o'-dicarbonic,3-methylindene-2-carboxy and 2-ethoxy-1-naphthoic acid.

Of the araliphatic acids there can be mentioned those with only onebenzene ring, optionally substituted as described previously, and inwhich the aliphatic chain preferably has from 1 to 6 carbon atoms, Suchacids can be straight-chained or branched, saturated or unsaturated, andcan also be substituted by one of the aforesaid functions or theirderivatives and/or can be interrupted by heteroatoms chosen from thegroup formed by N, O and S, or by other aromatic or heterocyclic oralicyclic nuclei. Specific acids of this type are, for example,phenylacetic, hydrotropic, cinnamic, phenylpropiolic, piperic, mandelic,3-(4-fluoro-benzoyl)-propionic, α-fluorocinnamic, 4-fluorocinnamic,3-fluoro-4-hydroxyphenylacetic, 4-fluorophenoxyacetic,α-fluorophenylacetic, 4-hydroxymandelic,(+)-6-methoxy-α-methyl-2-naphthalinacetic, 1-naphthoxyacetic,phenoxyacetic, 4-phenoxybenzoic, 3-trifluoromethylcinnamic,4-trifluoromethylmandelic, α,α,α-trifluoro-p-tolylacetic,3,4,5-trimethoxycinnamic, phenylglycine, D-4-hydroxyphenylglycine,α-sulfobenzeneacetic, 4-hydroxyphenylpropandioic,α-amino3,4-di-hydroxybenzeneacetic, 4-aminocinnamic,N-benzoyl-L-threonine, benzylthioglycolic, 4-bromomandelic,chloroacetyltyrosine, 2-chloro-6-fluorophenylacetic,4-chlorophenoxyacetic, transcinnamic, 3-(4-fluorobenzoyl)-propionic,4-fluorophenylacetic, DL-4-hydroxymandelic,2-(4-hydroxyphenoxy)-propionic, (S)-(+)-α-methoxyphenylacetic,(R)-(+)-α-methoxy-α-(trifluoromethyl)-phenylacetic, and(S)-(-)-α-methoxy-α-(trifluoromethyl)-phenylacetic acid.

Alicyclic acyl groups are primarily derived from acids containing from 1to 3 alicyclic rings, chosen preferably from those having 5 to 7 cycliccarbon atoms, optionally substituted by aromatic hydrocarbyl residues,for example benzene or naphthalene, or aliphatic residues, for examplealkyl or alkenyl, with preferably from 1 to 6 carbon atoms, or byhydroxy, oxo, amino or carboxy groups, free or functionally modified,for example as described previously. In those groups derived fromalicyclic acids as such, the carboxyl directly substitutes one or moreatoms of the ring hydrogens, or it may be found in one of the aforesaidaliphatic hydrocarbyl groups, thus providing alicyclic-aliphatic acids.In this case the aliphatic chain of such alicyclic aliphatic acids canbe substituted by functions such as those listed above for the case ofaraliphatic acids, or it can be interrupted by heteroatoms such as thosementioned previously. Acids specific to this series are, for example,cyclopropanecarboxy, cyclobutanecarboxy, cyclohexanecarboxy,1-amino-1cyclohexanecarboxy, cyclo-pentanecarboxy, 2,2-dichloro-1-methylcyclopropane-carboxy, 1-methyl-1-cyclohexanecarboxy,3-nor-adamantanecarboxy, 1-phenyl-1-cyclopropanecarboxy,(±)-1-benzocyclobutenecarboxy, (1S)-(-)-camphanic, (+)-camphorcarboxy,(-)-isoborneolacetic, (-)-menthoxy-acetic, 5-methoxy-1-indanon-3-acetic,3-methyl-1-adamantaneacetic, 3-methylinden-2-carboxy,2-nor-bornaneacetic, 1,2,3,4-tetrahydro-2-naphthoic, 1-adamantaneacetic,cycloheptanecarboxy, and cyclohexanebutyric acid.

One group of particular interest for the purposes of the presentinvention is comprised of steroid acids, such as for example cholic andcholanic acids, such as cholanic acid, cholic acid, lithocholic acid,deoxycholic acid and the respective ethio-acids, the ethio-acids derivedfrom androstane or pregnane or from their unsaturated derivatives in the4,5-position.

The heterocyclic residues of the acyls derived from acids of this seriescan be tricyclic or octacyclic, preferably between penta- andheptacyclic and can contain between one and four heteroatoms chosen fromthe group formed by O, N, and S and can be saturated or unsaturated, inparticular with an aromatic system of double bonds. Moreover, they canbe substituted by one or more of the groups already named with regard tothe aromatic and alicyclic acyl groups. In particular they can besubstituted by aliphatic hydrocarbyl groups, especially by alkyl groupswith a maximum of 6 carbon atoms, which can also be interrupted in thecarbon atom chain by one of the aforesaid heteroatoms. The heterocyclicgroups of this class can also be substituted with aliphatic, alicyclicor araliphatic acids and in this case too the aliphatic chain can besubstituted by one of the aforesaid groups, such as amino, hydroxy, freeor functionally modified sulfonic or sulfamide groups, or they can beinterrupted by other heteroatoms in the aforesaid manner.

Particular mention should be made of acyls derived from heterocyclicsingle-ringed acids, such as pyrrole, pyrazol, imidazol, thiophene,furan, pyran, pyridine, pyrimidine, pyrazine, thiopyran, oxazol,isoxazol, thiazole, isothiazole, triazols, tetrazol, triazines and thoseresulting from the condensation of these heterocyclics with a benzene ornaphthalic ring, or with several aromatic rings of this type, such asespecially indole, indolizine, coumarine, thionaphthene, carbazol,indazol, benzimidazol, benzothiazole, benzoisothiazole, quinoline,isoquinoline, acridine, phenanthridine, chromene, cinnoline,phthalazine, quinazoline, phenazine, phenoxazine, phenothiazine andbenzodiazepine, and those derived from the condensation of one or moreof the aforesaid heterocyclic compounds with other heterocyclics and/orwith aromatic or benzene rings.

Finally, acids of the aforesaid types derived from alkaloids are to beconsidered. Such acids are preferably known acids of biological ortherapeutic-pharmaceutical interest.

The acyls of the sphingosine and/or neuraminic groups according to thepresent invention can be derived from acid compounds of the aforesaidheterocyclic type, in which there are present double bonds of thearomatic type, such as in pyridine or pyrrole, or from correspondingderivatives, partially or completely hydrogenated, such as piperidine orpiperazine. The following acids are specific examples of such acids:2-furoic acid, 3-furoic acid, 2-thiopheneacetic acid,2-amino-4-thiazolacetic acid, nicotinic acid, isonicotinic acid,picolinic acid, 7-theophyllineacetic acid, 2-aminonicotinic acid,6-aminonicotinic acid, 5-aminoorotic acid, (S)-(-)-2-azetidincarboxyacid, 5-bromonicotinic acid, 5-chloroindol-2-carboxy acid,6-chloronicotinic acid, cinnoline-4-carboxy acid, L-histidine,N-acetyl-L-histidine, N-acetyl-L-tryptophan, 3-amino-4pyrazolcarboxyacid, 3-amino-1,2,4-triazol-5-carboxy acid, 5-benzimidazolcarboxy acid,2-benzofurancarboxy acid, (+)-biotin, 2-chloronicotinic acid,2,4-dihydroxypyrimidin-5-carboxy acid, 5-fluoroindol-2carboxy acid,2-furanpropionic acid, 5-hydantoinacetic acid, 5-hydroxyindol-3-aceticacid, 5-hydroxy-2-indolcarboxy acid, 6-hydroxynicotinic acid,4-imidazolacetic acid, 5-methoxyindole-3-acetic acid,5-methoxyindole-2-carboxy acid, 5-methoxy-2-methyl-3-indoleacetic acid,4-methoxy-2-quinolinecarboxy acid, kinurenic acid, thiokinurenic acid,7-chlorokinurenic acid, chlorothiokinurenic acids, fluorothiokinurenicacid and trifluoromethylthiokinurenic acid, 1-methylindol-2-carboxyacid, 6-methylnicotinic acid, N-methyl-L-proline acid,1-methyl-2-pyrrolcarboxy acid, 3-methyl-2-thiophenecarboxy acid,5-methyl-2-thiophenecarboxy acid, niflumic acid, 5-nitro-2-furoic acid,(-)-2-oxo-4-thiazolidincarboxy acid, 1-piperidinpropionic acid,2-pyrazincarboxy acid, 4-pyrazolcarboxy acid, 4-pyridazincarboxy acid,2-pyridylacetic acid, 3-(3-pyridyl)-acrylic acid, 4-pyridylthioaceticacid, (2-pyrimidylthio)acetic acid, quinaldicacid, 3-quinolincarboxyacid, 4-quinolincarboxy acid, 4-(2-thienyl)-butyric acid,3-thiopheneacetic acid, 2-thiopheneacetic acid, 2-(methylthio)-nicotinicacid, pyridylthioacetic acid, tetrazol-1-acetic acid, α-oxo-2furanaceticacid, (methoxymino)-2-furanacetic acid,2-α-(methoxymino)-4-thiazolacetic acid,α-[[(4-ethyl-2,3-dioxo-1-piperazinyl)carbonyl]amino]-benzeneacetic acid,1,3-dithiane-2-carboxy acid, 3-(2-chlorophenyl)-5-methyl-4-isoxazolcarboxy acid,3-(2-chloro-6-fluoro-phenyl)-5-methyl-4-isoxazolcarboxy acid, and 3-(2,6-dichlorophenyl)-4-isoxazolcarboxy acid.

In the N- and N'-acyl-N,N'-di-lysogangliosides according to the presentinvention, the acyl group is one of the aforesaid groups of thearomatic, alicyclic, araliphatic or heterocyclic series. Therefore, atleast one of the acyl groups, both on the sphingosine nitrogen and onthe neuraminic nitrogen, must be of this nature. In theN,N'-diacyl-N,N'-di-lysogangliosides, both the acyl groups can bederived from acids of the aforesaid series and such compounds are ofparticular importance with regard to the present invention since theyare easier to prepare.

In the N,N'-diacyl derivatives, one of the acyl groups can however alsobe derived from a saturated or unsaturated aliphatic acid, substitutedor not substituted, preferably with from 1 to 24 carbon atoms. Of suchacids can be mentioned the lower acids having from 1 to 11 carbon atoms,straight-chained or branched, such as formic acid, acetic acid,propionic acid, the butyric acids, the valerianic acids such asespecially n-valerianic acid and isovalerianic acid, pivalic acid,capronic and isocapronic acid, enanthic acid, caprylic acid, pelargonicacid, caprinic and undecylic acid, di-tert-butylacetic acid, and2-propylvalerianic acid. Of the unsaturated acids can be mentionedangelic acid and tiglic acid. Suitable longer-chained acids includethose with straight chains and especially those having from 12 to 16carbon atoms, for example lauric acid, myristic acid and palmitic acid.Those with an even higher carbon content include, for example, oleicacid, elaidinic acid, stearic acid, eicosancarbonic acid and behenicacid. In the acyl groups with branched chains, the lateral chains arepreferably lower alkyls with a maximum of 4 carbon atoms, especiallymethyl groups.

Of particular interest are N,N'-diacyl-N,N'-dilysogangliosides in whichan aliphatic acyl on the neuraminic nitrogen is a mixed acyl as ispresent in natural gangliosides, that is, acetyl for the most part andglycolyl for the lesser part, and wherein the hydroxyls of theneuraminic residue are also optionally acylated. Such derivatives areobtained by selective hydrolysis on the sphingosine nitrogen ofgangliosides and by acylation of the N'-lysogangliosides thus obtainedin the N-position with one of the aforesaid acids of the aromatic,araliphatic, alicyclic or heterocyclic series. Similarly, it is possibleto selectively hydrolyze the gangliosides on the neuraminic nitrogen andin the N'-lyso-gangliosides obtained to acylate the amino group in thisposition with one of the aforesaid non-aliphatic acids. The mixed acylsderived from higher fatty acids, as are present in natural gangliosides,remain on the sphingosine nitrogen. This is a preferred objective of thepresent invention.

The aliphatic acyl groups optionally present on the neuraminic nitrogenor sphingosine nitrogen can also be substituted by free functional orfunctionally modified groups, preferably functional polar groups.Preferably, from 1 to 3 functional groups are present and are chosenfrom the group formed by hydroxy, amino, ketone, mercapto, carboxy,sulfonic, sulfamide, sulfoxide, or sulfone and nitryl or nitro groupsand from the functional derivatives of these groups such as esters ofhydroxy, mercapto, carboxy, sulfonic, ketal, acetal, ketoxime, aldoxyand hydrazone groups. Groups of this type may be optionally substitutedwith lower aliphatic or araliphatic hydrocarbyl groups having from 1 to6 carbon atoms in the aliphatic part and preferably only one benzenering, such as alkylamine, alkyleneamine, alkylmercapto, alkylsulfamideand alkylhydrazone groups. Of the esters of hydroxyl groups can bementioned particularly those of the inorganic hydracids, that ishalogens, particularly chlorine, fluorine and bromine.

Of particular importance among the new acyl-di-lysogangliosides of theinvention are the N-acyl-N-lysogangliosides derived from naturalgangliosides, such as the gangliosides GM₁, GD_(1a), GD_(1b), GT_(1b),GM₂ and GM₃. In such derivatives the neuraminic acyl groups are thosewhich are present in such gangliosides, i.e., a mixed acetyl-glycolgroup, the acetyl group being prevalent, and in which the sialic hydroxygroups are optionally esterified with the corresponding acids. They areobtained from gangliosides by enzymatic hydrolysis involving adeacylation on the sphingosine nitrogen alone and by subsequentacylation with an aromatic, araliphatic, alicyclic or heterocyclic acid.The following are examples of such compounds:

N-2,6-dimethoxybenzoyl-N-lyso GM₁

N-5-methoxy-indanon-3-acetyl-N-lyso GM₁

N-phenylacetyl-N-lyso GM₁

N-cyclobutanecarbonyl-N-lyso GM₁

N-2-norbornaneacetyl-N-lyso GM₁

N-furoyl-N-lyso GM₁

N-imidazolacetyl-GM₁

N-6-methylnicotinyl-N-lyso GM₁

N-methylprolyl-N-lyso GM₁

N-1-methyl-2-pyrrolcarbonyl-N-lyso GM₁

N-2-pyridylacetyl-N-lyso GM₁

N-4,4-pyridylthioacetyl-N-lyso GM₁

N-3-quinolincarbonyl-N-lyso GM₁

N-tetrazolyl-l-acetyl-N-lyso GM₁

N-7-theophyllineacetyl-N-lyso GM₁

N-2-thiophenacetyl-N-lyso GM₁

N-3-amino-l,2,4,-triazol-5-acetyl-N-lyso GM₁

N-acetyl-DL-tryptophenacetyl-(α, α, α-trifluorotoluidin)-nicotinyl-GM₁

N-5-hydantoinacetyl-N-lyso GM₁

N-5-hydroxyindol-3-acetyl-N-lyso GM₁

N-2-chloronicotinyl-N-lyso GM₁

N-5-methyl-2-thiophenacetyl-N-lyso GM₁

N-5-benzimidazolacetyl-N-lyso GM₁

N-5-hydroxy-2-indolacetyl-N-lyso GM₁

N-3,4,5-trimethoxybenzoyl-N-lyso GM₁

N-cycloheptaneacetyl-N-lyso GM₁

N-cyclopentaneacetyl-N-lyso GM₁

N-5-methyl-2-thiophenacetyl-lyso GM₁ and the corresponding derivativesof the other basic gangliosides named previously and the inner esters ofall of these compounds.

Examples of N,N'-diacyl-N,N'-di-lysogangliosides are the derivativescorresponding to the aforesaid N-acyl-N-lysogangliosides derived fromGM₁ gangliosides or the other gangliosides, previously named, in whichthe amino group on the neuraminic nitrogen is also acylated with thesame acids, for example

N,N'-di-cycloheptylacetyl-N,N'-di-lyso GM₁,

N,N'-di-cyclopentylcarboxyl-N,N'-di-lyso GM₁,

N,N'-di-phenylacetyl-N,N'-dilyso GM₁, N,N'-di-pyridylacetyl-N,N'-di-lysoGM₁ or N,N'-di(5-methyl-2-thiophenacetyl)-di-lyso GM₁.

Examples of N'-monoacyl-N,N'-di-lysogangliosides derived from theganglioside GM₁ and from the other basic gangliosides named hereincontain the acyl groups which were mentioned in the examples specific toGM₁ N-mono-lysogangliosides on the neuraminic nitrogen instead of on thesphingosine nitrogen. Another interesting group of compounds accordingto the present invention are for example the ganglioside derivativescorresponding to the aforesaid N-acyl-N-lyso GM₁ compounds in which the"natural" mixed acyl on the neuraminic nitrogen is substituted by analiphatic acid with between 3 and 6 carbon atoms, such as valerianic orpivalic acid, or an acid of this type substituted with halogens, thatis, monochloroacetic or dichloroacetic acid, or an aliphatic acid withbetween 12 and 18 carbon atoms such as palmitic, oleic or stearic acid.

Of the compounds with functionally modified sialic carboxy functions canbe mentioned the esters derived from lower aliphatic alcohols havingfrom 1 to 6 carbon atoms, such as methyl, ethyl or propyl esters, theamides derived from lower aliphatic amines, such as methylamine,ethylamine or propylamine or cyclic amines, such as piperidine orpiperazine, pyrrolidine, the inner esters and the peracylates,peracetylates, perpropionylates, and perbutyrlates (that is, acylatesderived from aliphatic acids having from 1 to 6 carbon atoms) of all theaforesaid specific compounds. The semisynthetic ganglioside analogues ofthe present invention can be prepared in the known way, by acylating thedi-lysogangliosides or their N-acyl or N'-acyl derivatives, oroptionally selectively deacylating theN,N'-diacyl-N,N'-di-lysogangliosides on the sphingosine nitrogen and onthe neuraminic nitrogen.

In order to prepare di-acyl derivatives in which the acylamino groupsare derived from the same acid, it is preferable, for the sake ofsimplicity, to acylate the di-lysogangliosides in one single operationby the known procedures. The di-lysogangliosides can be obtained fromgangliosides or from N-lysogangliosides by alkaline hydrolysis, forexample with hydroxides of tetraalkylammonium, potassium hydroxide andothers. To prepare products according to the invention in which theacylamino groups are derived from different acids, it is preferable touse as starting compounds the N- or the N'-monoacyl derivatives ofdi-lysogangliosides. The N-mono-acyl-di-lysogangliosides can be obtainedby selective acylation from di-lysogangliosides, since the sphingosineamino group is more reactive than the neuraminic amino. Mild acylationof the dilysogangliosides according to known methods, for example by theacylation methods used in peptide chemistry, makes it possible to obtainthe aforesaid monoacyl derivatives on the sphingosine nitrogen. This isfollowed by acylation on the neuraminic nitrogen in the conventionalmanner. The acylation procedure to obtain the products according to theinvention consists in this case in a two-step acylation reaction.

Various methods can be used to prepare compounds with monoacyls derivedon the neuraminic nitrogen. It is possible, for example, to start fromdi-lysogangliosides and proceed to perform intermediate provisionalprotection of the sphingosine amino group, for example by hydrophobicinteraction with phosphatidylcholine, or by acylation with suitableprotecting groups, subsequent acylation on the neuraminic nitrogen witha derivative of the acid to be introduced in this position, thendeprotection on the sphingosine nitrogen. It is also possible to acylatethe di-lysogangliosides on the two amino groups with the same acid andthen expose the diacyl compound to the action of enzymes capable ofselectively splitting only the acylamino group on the sphingosinenitrogen, for example with enzymes used to obtain the lysogangliosidesfrom gangliosides. An example is theglycosphingolipid-ceramide-deacylase enzyme (see Scheme 1). ##STR6##N-monoacyl-N,N'-di-lysogangliosides can however also be obtained bydeacylation of N,N'-diacyl-N,N'-dilysogangliosides on the neuraminicnitrogen by selective chemical hydrolysis, for example with 0.1 molaralcoholic potassium hydroxide.

In the acyl-di-lysogangliosides obtained, it is possible, if desired, tofunctionally convert the carboxy groups of the sialic acids or hydroxylsof such acids. For example, these groups may be converted into esters oramides or the hydroxyls in these groups may be esterified with acids(peracylates).

The procedure for the preparation of N-acyl-N,N'-di-lyso-gangliosides,N'-acyl-N,N'-di-lysogangliosides andN,N'-diacyl-N,N'-di-lysogangliosides according to the present inventioncomprises acylating the N,N'-dilysogangliosides,N-acyl-N,N'-dilysogangliosides or N'-acyl-N,N'-di-lysogangliosides withthe acids corresponding to the aforesaid acyl groups or deacylating thesuitable N,N'-diacyl-N,N'-diacyl-N,N'-dilysogangliosides selectively onthe sphingosine nitrogen or on the neuraminic nitrogen, or mixtures ofthese compounds. If desired, the compounds obtained may be convertedinto esters, amides or inner esters or into hydroxy peracylates. Suchcompounds may also be converted into suitable salts.

The N-acylation according to the aforesaid procedure can be effected inthe conventional manner, for example by reacting the starting productswith an acylating agent, and above all with a functional derivative ofthe acid, the residue of which is to be introduced. Thus, it is possibleto use as the functional derivative of the acid a halogenide or ananhydride and acylation is then performed preferably in the presence ofa tertiary base, such as pyridine or collidine. It is possible tooperate under anhydrous conditions, at room temperature or higher oralso, to advantage, according to the method of Schotten-Baumann underaqueous conditions in the presence of an inorganic base. In some casesit is possible to also use the esters of the acids as reactivefunctional derivatives. To acylate, it is possible to use methods withactivated carboxy derivatives, such as are used in peptide chemistry,for example the method with mixed anhydrides or derivatives obtainablewith derivatives of carbodiimides or the salts of isoxazolium. Of thevarious preparation methods, the most appropriate are the following:

1. Reaction of the lysoganglioside derivative with the azide of theacid;

2. Reaction of the lysoganglioside derivative with an acylimidazole ofthe acid obtainable from the acid with N,N'-carbonyldiimidazole;

3. Reaction of the lysoganglioside derivative with a mixed anhydride ofthe acid and trifluoroacetic acid;

4. Reaction of the lysoganglioside derivative with the acid chloride;

5. Reaction of the lysoganglioside derivative with the acid in thepresence of a carbodiimide (such as dicyclohexylcarbodiimide) andoptionally of a substance such as 1-hydroxy-benzotriazol;

6. Reaction of the lysoganglioside derivative with the acid by heating;

7. Reaction of the lysoganglioside derivative with a methyl ester of theacid at a high temperature;

8. Reaction of the lysoganglioside derivative with a phenol ester of theacid, such as an ester with para-nitrophenol; and

9. Reaction of the lysoganglioside derivative with an ester derived fromthe exchange between a salt of the acid and 1-methyl-2-chloropyridiniumiodide.

It has already been noted how it is possible to obtain selective partialacylation both on the sphingosine nitrogen and on the neuraminicnitrogen. Scheme 1 illustrates the procedures.

Enzymatic deacylation of N,N'-diacyl-N,N'-dilysogangliosides on thesphingosine nitrogen as reported previously can be carried out under theconditions used for the partial deacylation of gangliosides, for exampleas described in J. Biochem., 103, 1 (1988). The double deacylation ofN,N'-diacyl-N,N'-di-lysogangliosides to N,N'-di-lysogangliosides can beeffected in the same way as for the preparation ofde-N-acetyl-lysogangliosides as described for example in Biochemistry24, 525 (1985); J. Biol. Chem. 255, 7657, (1980); Biol. Chem. HoppeSeyler 367, 241, (1986); Carbohydr. Research 179, 393 (1988); Bioch.Bioph. Res. Comn. 147, 127 (1987).

The aforesaid publication in Carbohydr. Research 179 also describes amethod for the selective deacylation on the neuraminic nitrogenobtainable by the action of KOH 0.1M in 90% n-butanol of the gangliosideGM₃. This deacylation can be applied toN,N'-diacyl-N,N'-dilysogangliosides of the present invention to obtainN-acyl-N,N'-di-lysogangliosides. Of course, the preparation methodscoming within the scope of the present invention also include anychemical equivalents apparent to one skilled in the art.

The preparation of carboxy or hydroxy derivatives of the novel acyllysogangliosides obtained according to the aforesaid procedure can beeffected by the known procedures, except those methods which would havethe effect of altering the basic ganglioside structure. This wouldexclude methods that employ highly acidic agents, or which would howeverbe carried out under drastically alkaline or acid conditions, or alsothose methods which would lead to an undesired alkylation of the hydroxygroups of the saccharide part.

The esterification of carboxy groups of the N-acyl gangliosides or theirconversion into amides can be effected for example as described in U.S.Pat. No. 4,713,374 for gangliosides. The formation of inner esters ofthe derivatives of the invention can be effected as in the case of thepreparation of inner esters of gangliosides, as described for example inU.S. Pat. No. 4,593,091 and in EP patent 0072 722.

These inner esters include not only the compounds formed bylactonization of sialic carboxy groups with saccharide hydroxyls, butalso those for example which contain lactone rings formed between thesialic carboxyls and the sialic hydroxyls, since the latter are in turnbound to the saccharide part, and also other possible lactonestructures. The procedure of the aforesaid patents for the formation ofinner esters comprises treating a ganglioside in a non-aqueous organicsolvent under anhydrous conditions with a lactonizing agent. Suitableorganic solvents include dimethylsulfoxide, dimethylformamide,sulfolane, tetrahydrofuran, dimethoxyethane, pyridine or mixtures ofthese solvents. Suitable reagents for lactonization includecarbodiimides soluble in organic solvents, such asdicyclohexylcarbodiimide, benzylisopropylcarbodiimide,benzylethylcarbodiimide, salts of 2-chloromethylpyridine,ethoxyacetylene and Woodward's reagent(N-ethyl-5-phenylisoxazolium-3'-sulfonate). Older methods make use ofthe reaction between a ganglioside and acetic acid or trichloroaceticacid or a carbodiimide, soluble in water or in an aqueous medium. Allthese methods can also be used to prepare inner esters of the new N-acyllysogangliosides. For the "outer" esterification of carboxy groups, thatis, esterification with alcohols of the aforesaid series, it is possiblefor example to react the N-acyl lysogangliosides with the desiredalcohol, in the presence of an ion exchanger, e.g. a Dowex 50-typeresin, the yield being limited by the simultaneous formation of inneresters and the reaction times being rather long. Another method ofesterification comprises passing the alcohol on a resin, of the Dowex-50Wx8 (100-200 mesh form H) type, and treating the dissolved eluate inthe same alcohol with the corresponding diazoalkane.

Another suitable ester preparation method comprises treating a metalsalt of the lysoganglioside derivative with an etherifying agent.Alkaline and alkaline earth metal salts may be used, but also any othermetal salt. As etherifying agents it is possible to use those reportedin the literature, such as especially the esters of various inorganicacids, or of organic sulfonic acids, such as hydracids, that is, inother words, the hydrocarbyl halogenides, such as methyl or ethyl iodideetc., or the neutral sulfates or acids of hydrocarbyls, sulfites,carbonates, silicates, phosphites or hydrocarbyl sulfonates, for examplemethyl benzo- or ptoluolsulfonate. The reaction can be effected in asuitable solvent, for example an alcohol, preferably the one whichcorresponds to the alkyl group to be introduced, but also in non-polarsolvents, such as ketones or ethers, such as dioxane ordimethylsulfoxide. One particularly advantageous method ofesterification comprises treating an inner ester of the lysogangliosidederivative with a mixture of the desired alcohol and its correspondingalcoholate. The reaction can be conducted at a temperature correspondingto the boiling point of the alcohol, but it is also possible to uselower temperatures, the reaction times in this case being longer.

The amides of the lysoganglioside derivatives of the present inventioncan be prepared by known methods, and especially by the following:

(a) Reaction of the inner esters of the N-acyl lysogangliosidederivatives with ammonia or with the amines;

(b) Reaction of the carboxy esters of the N-acyl lysogangliosidederivatives with ammonia or with the amines; and

(c) Reaction of the N-acyl lysoganglioside derivatives with the carboxygroups activated with ammonia or with the amines.

Reaction (a) can be effected by direct treatment, with or withoutsolvent, of the ganglioside inner ester with ammonia or with the amineof which the amide is to be prepared. The reaction can be effected alsoat quite low temperatures, such as -5° to +10° , but preferably at roomtemperature or higher, for example between 30° and 120°. As solvents, itis possible to use ketones, aromatic hydrocarbides, dimethylformamide,dimethylsulfoxide, dioxane or tetrahydrofuran. Reaction (b) ispreferably effected under the conditions described for reaction (a).Apart from the esters described for the present invention, it ispossible to also use other esters, for example esters with phenols.

To activate the carboxy group in the reaction according to (c), methodsknown in the field of peptide chemistry may be employed, avoiding thosewhich involve conditions that are too acidic or basic, which could leadto the disintegration of the ganglioside molecule. If the startinggangliosides are in the form of, for example, sodium salts, it isadvisable to first treat the salt with an ion exchange resin,Dowex-type, or another acid ion exchanger. It is possible to use themethod of condensation in the presence of carbodiimides, for exampledicyclohexylcarbodiimide, benzylisopropylcarbodiimide orbenzylethylcarbodiimide, in the presence of 1-hydroxybenzotriazol orcondensation in the presence of N,N'-carbonyldiimidazol.

Acylation of the hydroxy groups of the saccharide, sialic part andoptionally of the ceramide residue can also be carried out in the knownway, for example by acylation with a halogenide or an anhydride of theacid to be used for acylation, preferably in the presence of a tertiarybase, such as pyridine or collidine. As a result, the aforesaidperacylated derivatives are obtained. It is also possible, according tothe definition of the procedure of the present invention, to expose toacylation a de-N-acetyl lysoganglioside and to recover the acetylaminogroup in the neuraminic acid after acylation. Such acetylation can alsobe effected in the known way. In this case relatively mild methods arechosen for the N-acylation, by which the hydroxy group of the neuraminicacid remains unaltered. Acetylation of this group, to be effected afterthe acylation reaction on the sphingosine nitrogen, can be done bydrastic methods, for example, by using acetic anhydride.

Finally, as noted above, in all the compounds obtainable by theaforesaid procedure which present salifiable groups, it is possible tosalify such groups in the known way to obtain appropriate saltderivatives.

The invention also includes modifications of the preparation procedureof the new derivatives, in which a procedure is interrupted at any onestage or is started with an intermediate compound and the remainingsteps are performed, or in which the starting products are formed insitu.

Also included in the present invention are pharmaceutical preparationswhich contain as active substances one or more of the new acyllysoganglioside derivatives and, in particular, those mentioned herein.The pharmaceutical preparations mentioned herein can be preparations fororal, rectal, parenteral, local or transdermal use. They are thereforein solid or semisolid form, for example pills, tablets, gelatinouscapsules, capsules, suppositories, and soft gelatin capsules. Forparenteral use it is possible to use forms designed for intramuscular,subcutaneous or transdermal administration, or which are suitable forinfusions or intravenous injections. These preparations can therefore bein the form of solutions of the active compounds or as freeze-driedpowders of the active compounds to be mixed with one or morepharmaceutically acceptable excipients or diluents, convenient for theaforesaid uses and with an osmolarity that is compatible with thephysiological fluids. For local use, preparations in the form of sprays,for example nasal sprays, creams or ointments for topical use orsuitably prepared plasters for transdermal use can be used.

The preparations of the invention can be administered to humans oranimals. They contain preferably from 0.01% to 10% by weight of theactive compound for solutions, sprays, ointments and creams and from 1%to 100% and preferably from 5% to 50% by weight of active compound forpreparations in solid form. The dosage to be administered depends onindividual indications, on the desired effect and on the chosenadministration route. Another feature of the present invention isrepresented by the therapeutic use both of the new acyllysogangliosidesand of those which are already known and listed previously. Thistherapeutic use includes all of the previously listed indications. Thedaily dosages to man by injection (subcutaneous or intramuscular) ortransdermal or oral administration vary between 0.05 mg to 5 mg ofactive substance per kg of body weight. The following Examplesillustrate the preparation of the acyl-lysogangliosides of the presentinvention and the pharmaceutical preparations containing them as activeingredients, and their therapeutic uses. These Examples are merelyillustrative and are not to be considered as limiting of the presentinvention.

EXAMPLE 1 N,N'-di-lyso GM₁

10 g of GM₁ are dissolved in 200 ml KOH 3N and hydrolysis is effectedfor 72 hrs at 90° C.

The solution is then cooled and brought to pH 6.5 with hydrochloricacid. It is left to stand for 18 hrs at 4° C. and then the precipitatedfatty acids are filtered away. The resulting solution is dialyzedagainst water and concentrated to 500 ml and precipitated in 5 liters(lt) of acetone.

The product is dried and high performance silica gel chromatography iseffected using as eluent a mixture of chloroform/methanol/NH₃ 5N(55:45:10). The fractions containing the product are dried and thenredissolved in water. It is brought to pH 10 with NaOH 0.01N anddialyzed, concentrated to 100 mg/ml and precipitated in 5 volumes ofacetone. Yield of N,N'-di-lyso GM₁ is 5.7 g (70% theoretical). Silicagel chromatography with a solvent formed by chloroform/methanol/NH₃ 5N(55:45:10) shows the product to be a unitary compound with Rf=0.05 (GM₁=0.35) .

EXAMPLE 2 N-lyso GM₁

10 g (6.37 mM) of GM₁ are dissolved in 200 ml KOH 3N and hydrolysis iseffected for 72 hrs at 90° C. The solution is then cooled and brought topH 6.5 with hydrochloric acid. It is left to stand for 18 hrs at 4° C.and then the precipitated fatty acids are filtered away. It is dialyzedagainst water and concentrated to 500 ml and precipitated in 5 liters ofacetone.

The product containing N'-lyso GM₁ and N,N'-dilyso GM₁ (20%) isvacuum-dried and then redissolved in 100 ml of dimethylformamide. 2.15 g(6.37 mM) of 9-fluorenylmethyloxycarbonyl-N-hydroxysuccinimide dissolvedin 20 ml of tetrahydrofuran are then slowly added and left to react for1 hr at room temperature. Thereafter, 3 ml (31.85 mM) of aceticanhydride and 0.9 ml (63.7 mM) of triethylamine are added. After 30minutes, 12.5 ml of piperidine are added to remove the protecting group.The mixture is left to react for 18 hrs at room temperature andprecipitated in 2 liters of acetone and dried. The material thusobtained is dissolved in Na₂ CO₃ 1M and kept at 60° C. for 1 hr. It isdialyzed, concentrated to 100 mg/ml and precipitated in 5 volumes ofacetone.

The product is passed through an S Sepharose column (H⁺ form)equilibrated in methanol. It is washed with methanol and N-lyso GM₁ byeluting with NH₄ Cl 10 mM in methanol. The fractions containing theproduct are dried and then redissolved in water. The solution is broughtto pH 10 with NaOH 0.01N and dialyzed, concentrated to 100 mg/ml andprecipitated in 5 volumes of acetone.

Product obtained: approximately 5 g (60% theoretical).

Silica gel chromatography with a solvent formed bychloroform/methanol/NH₃ 5N (55:45:10) shows the product to be unitarywith Rf=0.11.

EXAMPLE 3 N-cyclobutanecarbonyl-N-lyso GM₁

500 mg (0.38 mM) of Lyso GM₁ (prepared according to Example 2) aredissolved in 1 ml of dimethylformamide, and 1.050 ml (7.6 mM) oftriethylamine and 728 μl (7.6 mM) of cyclobutanecarbonyl chloride areadded at room temperature.

The condensation reaction is conducted at room temperature for 4 hrs. Atthe end of the reaction, the solution is precipitated in 10 ml of ethylacetate saturated with water, filtered and vacuum-dried. The product isthen purified by silica gel chromatography using as eluent a mixture ofchloroform/methanol/water (60:35:8).

The pure fractions are pooled, evaporated, treated with Na₂ CO₃ 1N,dialyzed against distilled water and then concentrated to 5 ml, andprecipitated in 50 ml of acetone.

Product obtained: 350 mg (66% theoretical).

Silica gel chromatography with a solvent formed bychloroform/methanol/CaCl₂ 0.3% (50:42:11), shows the compound to beunitary with Rf=0.33 (GM₁ =0.43; Lyso GM₁ =0.24).

EXAMPLE 4 N-(2-norbornaneacetyl)-N-lyso GM₁

500 mg (0.38 mM) of Lyso GM₁ (prepared according to Example 2) aredissolved in 2.5 ml of dimethylformamide and then are added, at 0° C.,106 μl (0.76 mM) of triethylamine and norbornaneacetic anhydride freshlyprepared by reacting 1.1 ml (7.6 mM) of 2-norbornaneacetic acid and 939mg (9.12 mM) of dicyclohexylcarbodiimide dissolved in 20 ml oftetrahydrofuran and after filtering, 2 hrs later, the dicyclohexylureawhich has formed.

The condensation reaction is conducted at 0° C. for 18 hrs understirring. At the end of the reaction, the solution is concentrated to 1ml, precipitated in 10 ml of acetone and vacuum-dried.

The product is then purified by silica gel chromatography using aseluent a mixture of chloroform/methanol/water (60:35:8).

The pure fractions are pooled, evaporated, treated with Na₂ CO₃ 1N,dialyzed against distilled water and then concentrated to 5 ml andprecipitated in 50 ml of acetone.

Product obtained: 508 mg (92% theoretical).

Silica gel chromatography with a solvent formed bychloroform/methanol/CaCl₂ 0.3% (50:42:11), shows the compound to beunitary with Rf=0.33 (GM₁ =0.43; Lyso GM₁ =0.24).

EXAMPLE 5 N-phenylacetyl-N-lyso GM₁

500 mg (0.38 mM) of Lyso GM₁ (prepared according to Example 2) aredissolved in 2.5 ml of dimethylformamide after which are added, at roomtemperature, 528 μl (7.6 mM) of triethylamine, 260 mg (1.9 mM) ofphenylacetic acid and 194.2 mg (0.76 mM) of 1- methyl-2-chloropyridiniumiodide dissolved in 2.5 ml of dimethylformamide.

The mixture is left to react for 18 hrs at room temperature and thenprecipitated in 100 ml of acetone. It is filtered and dried. The productis then purified by silica gel chromatography using as eluent a mixtureof chloroform/methanol/water (60:35:8).

The pure fractions are pooled, evaporated, treated with Na₂ CO₃ 1N,dialyzed against distilled water and then concentrated to 5 ml andprecipitated in 50 ml of acetone.

Product obtained: 354 mg (65% theoretical).

Silica gel chromatography with a solvent formed bychloroform/methanol/CaCl₂ 0.3% (50:42:11), shows the compound to beunitary with Rf=0.37 (GM₁ =0.43; Lyso GM₁ =0.24) and with a fluorometricreading of 254 nm.

EXAMPLE 6 N-(2,6-dimethoxybenzoyl)-N-lyso GM₁

500 mg (0.38 mM) of lyso GM₁ (prepared according to Example 2) aredissolved in 1 ml of dimethylformamide, and then are added at roomtemperature 1,056 ml (7.6 mM) of triethylamine and dimethoxybenzoicanhydride freshly prepared by reacting 2.76 g (15.2 mM) of2,6-dimethoxybenzoic acid and 1.08 g (3.8 mM) of1-methyl-2-fluoropyridinium p-toluenesulfonate in 10 ml ofdimethylformamide/tetrahydrofuran 1:1.

The condensation reaction is conducted at room temperature for 4 hrsunder stirring. At the end of the reaction, the solution is concentratedto 5 ml, precipitated in 50 ml of acetone, filtered and vacuum-dried.Silica gel chromatography is effected, using as eluent a mixture ofchloroform/methanol/water (60:35:8). The pure fractions are pooled,evaporated, gathered with Na₂ CO₃ 1N, dialyzed against distilled waterand then concentrated to 5 ml and precipitated in 50 ml of acetone.

Product obtained: 506 mg (90% theoretical).

Silica gel chromatography with a solvent formed bychloroform/methanol/CaCl₂ 0.3% (50:42:11), shows the compound to beunitary with Rf=0.35 (GM₁ =0.43; Lyso GM₁ =0.24) and with a fluorometricreading of 254 nm.

EXAMPLE 7 N-(2-furoyl)-N-lyso GM₁

500 mg (0.38 mM) of Lyso GM₁ (prepared according to Example 2) aredissolved in 2.5 ml of dimethylformamide, after which are added, at roomtemperature, 528 μl (3.8 mM) of triethylamine, 210 mg (1.9 mM) of2-furoic acid and 194.2 mg (0.76 mM) of 1-methyl-2-chloropyridiniumiodide dissolved in 2.5 ml of dimethylformamide.

The mixture is left to react for 18 hrs at room temperature and thenprecipitated in 10 ml of acetone. It is filtered and dried. The productis then purified by silica gel chromatography using as eluent a mixtureof chloroform/methanol/water (60:35:8). The pure fractions are pooled,evaporated, treated with Na₂ CO₃ 1N, dialyzed against distilled waterand then concentrated to 5 ml and precipitated in 50 ml of acetone.

Product obtained: 402 mg (75% theoretical).

Silica gel chromatography with a solvent formed bychloroform/methanol/CaCl₂ 0.3% (50:42:11), shows the compound to beunitary with Rf=0.37 (GM₁ =0.43; Lyso GM₁ =0.24) and with a fluorometricreading of 254 nm.

EXAMPLE 8 N-(4-imidazolacetyl)-N-lyso GM₁

500 mg (0.38 mM) of Lyso GM₁ (prepared according to Example 2) aredissolved in 2.5 ml of dimethylformamide, after which are added, at roomtemperature, 1056 μl (7.6 mM) of triethylamine, 310 mg (1.9 mM) of4-imidazolacetic acid hydrochloride and 194.2 mg (0.76 mM) of1-methyl-2-chloropyridinium iodide dissolved in 2.5 ml ofdimethylformamide. It is left to react for 18 hrs at room temperatureand then precipitated in 100 ml of acetone, filtered and dried. Theproduct is then purified by silica gel chromatography using as eluent amixture of chloroform/methanol/water (60:35:8). The pure fractions arepooled, evaporated, treated with Na₂ CO₃ 1N, dialyzed against distilledwater and then concentrated to 5 ml and precipitated in 50 ml ofacetone.

Product obtained: 352 mg (65% theoretical).

Silica gel chromatography with a solvent formed bychloroform/methanol/CaCl₂ 0.3% (50:42:11), shows the compound to beunitary with Rf=0.33 (GM₁ =0.43; Lyso GM₁ =0.24).

EXAMPLE 9 N-(1-methylprolyl)-N-lyso GM₁

500 mg (0.38 mM) of Lyso GM₁ (prepared according to Example 2) aredissolved in 3.5 ml of dimethylformamide/water 2.5:1 after which areadded, at room temperature, 1056 μl (7.6 mM) of triethylamine, 260 mg(1.9 mM) of N-methyl-L-proline and 194.2 mg (0.76 mM) of1-methyl-2-chloropyridinium iodide dissolved in 2.5 ml ofdimethylformamide. The mixture is left to react for 18 hrs at roomtemperature and then precipitated in 100 ml of acetone, filtered anddried. The product is then purified by silica gel chromatography usingas eluent a mixture of chloroform/methanol/water (60:35:8). The purefractions are pooled, evaporated, treated with Na₂ CO₃ 1N, dialyzedagainst distilled water and then concentrated to 5 ml and precipitatedin 50 ml of acetone.

Product obtained: 380 mg (70% theoretical).

Silica gel chromatography with a solvent formed bychloroform/methanol/CaCl₂ 0.3% (50:42:11), shows the compound to beunitary with Rf=0.33 (GM₁ =0.43; Lyso GM₁ =0.24).

EXAMPLE 10 N-(1-methyl-2-pyrrolecarbonyl)-N-lyso GM₁

500 mg (0.38 mM) of Lyso GM₁ (prepared according to Example 2) aredissolved in 2.5 ml of dimethylformamide after which are added, at roomtemperature, 528 μl (7.6 mM) of triethylamine, 220 mg (1.9 mM) of1-methyl-2-pyrrolecarboxy acid and 194.2 mg (0.76 mM) of1-methyl-2-chloropyridinium iodide dissolved in 2.5 ml ofdimethylformamide.

It is left to react for 18 hrs at room temperature and then precipitatedin 100 ml of acetone. The reaction mixture is filtered and dried. Theproduct is then purified by silica gel chromatography using as eluent amixture of chloroform/methanol/water (60:35:8). The pure fractions arepooled, evaporated, treated with Nahd 2CO₃ 1N, dialyzed againstdistilled water and then concentrated to 5 ml and precipitated in 50 mlof acetone.

Product obtained: 487 mg (90% theoretical).

Silica gel chromatography with a solvent formed bychloroform/methanol/CaCl₂ 0.3% (50:42:11), shows the compound to beunitary with Rf=0.38 (GM₁ =0.43; Lyso GM₁ =0.24) and with a fluorometricreading of 254 nm.

EXAMPLE 11 N-(1-tetrazolacetyl)-N-lyso GM₁

500 mg (0.38 mM) of Lyso GM₁ (prepared according to Example 2) aredissolved in 2.5 ml of dimethylformamide after which are added, at roomtemperature, 528 μl (7.6 mM) of triethylamine, 250 mg (1.9 mM) of1-tetrazolacetic acid and 194.2 mg (0.76 mM) of1-methyl-2-chloropyridinium iodide dissolved in 2.5 ml ofdimethylformamide.

The mixture is left to react for 18 hrs at room temperature and thenprecipitated in 100 ml of acetone. It is filtered and dried. The productis then purified by silica gel chromatography using as eluent a mixtureof chloroform/methanol/water (60:35:8). The pure fractions are pooled,evaporated, treated with Na₂ CO₃ 1N, dialyzed against distilled waterand then concentrated to 5 ml and precipitated in 50 ml of acetone.

Product obtained: 472 mg (87% theoretical).

Silica gel chromatography with a solvent formed bychloroform/methanol/CaCl₂ 0.3% (50:42:11), shows the compound to beunitary with Rf=0.32 (GM₁ =0.43; Lyso GM₁ =0.24).

EXAMPLE 12 N-(2-thiopheneacetyl)-N-lyso GM₁

500 mg (0.38 mM) of Lyso GM₁ (prepared according to Example 2) aredissolved in 2.5 ml of dimethylformamide after which are added, at roomtemperature, 528 μl (7.6 mM) of triethylamine, 270 mg (1.9 mM) of2-thiopheneacetic acid and 194.2 mg (0.76 mM) of 1-methyl-2-chloropyridinium iodide dissolved in 2.5 ml of dimethylformamide.

It is left to react for 18 hrs at room temperature and then precipitatedin 100 ml of acetone, filtered and dried. The product is then purifiedby silica gel chromatography using as eluent a mixture ofchloroform/methanol/water (60:35:8). The pure fractions are pooled,evaporated, treated with Na₂ CO₃ 1N, dialyzed against distilled waterand then concentrated to 5 ml and precipitated in 50 ml of acetone.

Product obtained: 383 mg (70% theoretical).

Silica gel chromatography with a solvent formed bychloroform/methanol/CaCl₂ 0.3% (50: 42: 11) , shows the compound to beunitary with Rf=0.34 (GM₁ =0.43; Lyso GM₁ =0.24) and with a fluorometricreading of 254 nm.

EXAMPLE 13 N-(6-methylnicotinoyl)-N-lyso GM₁

500 mg (0.38 mM) of Lyso GM₁ (prepared according to Example 2) aredissolved in 2.5 ml of dimethylformamide after which are added, at roomtemperature, 528 μl (7.6 mM) of triethylamine, 260 mg (1.9 mM) of6-methylnicotinic acid and 194.2 mg (0.76 mM) of1-methyl-2-chloropyridinium iodide dissolved in 2.5 ml ofdimethylformamide.

The mixture is left to react-for 18 hrs at room temperature and thenprecipitated in 100 ml of acetone. It is filtered and dried. The productis then purified by silica gel chromatography using as eluent a mixtureof chloroform/methanol/water (60:35:8).

The pure fractions are pooled, evaporated, treated with Na₂ CO₃ 1N,dialyzed against distilled water and then concentrated to 5 ml andprecipitated in 50 ml of acetone.

Product obtained: 218 mg (40% theoretical).

Silica gel chromatography with a solvent formed bychloroform/methanol/CaCl₂ 0.3% (50:42:11), shows the compound to beunitary with Rf=0.38 (GM₁ =0.43; Lyso GM₁ =0.24) and with a fluorometricreading of 254 nm.

EXAMPLE 14 N-(2-pyridylacetyl)-N-lyso GM₁

500 mg (0.38 mM) of Lyso GM₁ (prepared according to Example 2) aredissolved in 3.5 ml of dimethylformamide/water 2.5:1 after which areadded, at room temperature, 1056 μl (7.6 mM) of triethylamine, 330 mg(1.9 mM) of 2-pyridyl-acetic acid hydrochloride and 194.2 mg (0.76 mM)of 1-methyl-2-chloropyridinium iodide dissolved in 2.5 ml ofdimethylformamide.

It is left to react for 18 hrs at room temperature and then precipitatedin 100 ml of acetone, filtered and dried. The product is then purifiedby silica gel chromatography using as eluent a mixture ofchloroform/methanol/water (60:35:8). The pure fractions are pooled,evaporated, treated with Nahd 2CO₃ 1N, dialyzed against distilled waterand then concentrated to 5 ml and precipitated in 50 ml of acetone.

Product obtained: 327 mg (60% theoretical).

Silica gel chromatography with a solvent formed bychloroform/methanol/CaCl₂ 0.3% (50:42:11), shows the compound to beunitary with Rf=0.35 (GM₁ =0.43; Lyso GM₁ =0.24) and with a fluorometricreading of 254 nm.

EXAMPLE 15 N-(4-pyridylthioacetyl)-N-lyso GM₁

500 mg (0.38 mM) of Lyso GM₁ (prepared according to Example 2) aredissolved in 2.5 ml of dimethylformamide after which are added at roomtemperature 528 μl (7.6 mM) of triethylamine, 320 mg (1.9 mM) of4-pyridylthioacetic acid and 194.2 mg (0.76 mM) of1-methyl-2-chloropyridinium iodide dissolved in 2.5 ml ofdimethylformamide.

It is left to react for 18 hrs at room temperature and then precipitatedin 100 ml of acetone, filtered and dried. The product is then purifiedby silica gel chromatography using as eluent a mixture ofchloroform/methanol/water (60:35:8).

The pure fractions are pooled, evaporated, treated with Na₂ CO₃ 1N,dialyzed against distilled water and then concentrated to 5 ml andprecipitated in 50 ml of acetone.

Product obtained: 390 mg (70% theoretical).

Silica gel chromatography with a solvent formed bychloroform/methanol/CaCl₂ 0.3% (50:42:11), shows the compound to beunitary with Rf=0.34 (GM₁ =0.43; Lyso GM₁ =0.24) and with a fluorometricreading of 254 nm.

EXAMPLE 16 N-(3-quinolincarbonyl)-N-lyso GM₁

500 mg (0.38 mM) of Lyso GM₁ (prepared according to Example 2) aredissolved in 2.5 ml of dimethylformamide after which are added at roomtemperature 528 μl (7.6 mM) of triethylamine, 330 mg (1.9 mM) of3-quinolincarboxy acid and 194.2 mg (0.76 mM) of1-methyl-2-chloropyridinium iodide dissolved in 2.5 ml ofdimethylformamide.

The mixture is left to react for 18 hrs at room temperature and thenprecipitated in 100 ml of acetone, filtered and dried. The product isthen purified by silica gel chromatography using as eluent a mixture ofchloroform/methanol/water (60:35:8).

The pure fractions are pooled, evaporated, treated with Na₂ CO₃ 1N,dialyzed against distilled water and then concentrated to 5 ml andprecipitated in 50 ml of acetone.

Product obtained: 358 mg (64% theoretical).

Silica gel chromatography with a solvent formed by chloroformmethanol/CaCl₂ 0.3% (50:42:11), shows the compound to be unitary withRf=0.35 (GM₁ =0.43; Lyso GM₁ =0.24) and with a fluorometric reading of254 nm.

EXAMPLE 17 N-(7-theopbyllinacetyl)-N-lyso GM₁

500 mg (0.38 mM) of Lyso GM₁ (prepared according to Example 2) aredissolved in 2.5 ml of dimethylformamide after which are added at roomtemperature 528 μl (7.6 mM) of triethylamine, 460 mg (1.9 mM) of7-theophyllineacetic acid and 194.2 mg (0.76 mM) of1-methyl-2-chloropyridinium iodide dissolved in 2.5 ml ofdimethylformamide.

It is left to react for 18 hrs at room temperature and then precipitatedin 100 ml of acetone, filtered and dried. The product is then purifiedby silica gel chromatography using as eluent a mixture ofchloroform/methanol/water (60:35:8).

The pure fractions are pooled, evaporated, treated with Na₂ CO₃ 1N,dialyzed against distilled water and then concentrated to 5 ml andprecipitated in 50 ml of acetone.

Product obtained: 397 mg (68% theoretical).

Silica gel chromatography with a solvent formed bychloroform/methanol/CaCl₂ 0.3% (50:42:11), shows the compound to beunitary with Rf=0.35 (GM₁ =0.43; Lyso GM₁ =0.24) and with a fluorometricreading of 254 nm.

EXAMPLE 18 N-2,6-dimethoxybenzoyl-di-lyso GM₁

500 mg of di-lyso GM₁ (0.39 mM) (prepared according to Example 1) aredissolved in 5 ml of dimethylformamide, and to this solution are thenslowly added 145 mg (0.43 mM) of9-fluorenylmethyl-oxycarbonyl-N-hydroxysuccinimide (FMOC-succ.). It isleft to react for 1 hr at room temperature.

At the end of the reaction, it is precipitated in 100 ml of acetone,filtered and dried. The product is then purified by silica gelchromatography using as elution solvent a mixture ofchloroform/methanol/water 60:30:6.

The fractions containing the intermediate reaction productN-FMOC-di-lyso-GM₁ are pooled, dried and then redissolved in 2.5 ml ofdimethylformamide methanol 1:1 after which are added, at 0° C., 1.1 ml(7.92 mM) of triethylamine and 0.40 ml (3.96 mM) of methyltrifluoroacetate. It is left to react at room temperature for 3 days.

To the resulting reaction mixture is then added 1 ml of piperidine toremove the fluorenyl group. It is left to react for 18 hrs at roomtemperature and precipitated in 100 ml of acetone/water 9:1, filteredand dried.

The intermediate reaction product is dissolved in 2.5 ml ofdimethylformamide/methanol 1:1 and to this is added dimethoxybenzoicanhydride, freshly prepared by reacting 1.88 g (7.6 mM) of2,6-di-methoxybenzoic acid and 5.40 mg (1.9 mM) offluoromethylpyridinium para-toluenesulfonate in 5 ml ofdimethylformamide/tetrahydrofuran 1:1. This mixture is left to react for18 hrs at room temperature, precipitated in 100 ml of acetone, gatheredwith 5 ml of water and brought to pH 9.0 with NaOH 0.01N.

It is left to react at room temperature for 2 hrs to remove thetrifluoroacetyl group. It is dialyzed, concentrated to 3 ml andprecipitated in 15 ml of acetone.

The raw product obtained is purified by silica gel chromatography usingas eluent a mixture of chloroform/methanol/water 60:35:8.

The pure fractions are pooled, evaporated, redissolved in 2 ml ofchloroform/methanol 1:1 and precipitated in 10 ml of acetone.

Product obtained=283 mg (51% theoretical)

Silica gel chromatography with a solvent formed bychloroform/methanol/CaCl₂ 0.3% 50:42:11, shows the compound to beunitary with Rf=0.18. It proves positive to ninhydrin staining and showsa fluorometric reading of 254 nm.

EXAMPLE 19 N-2-pyridylacetyl-di-lyso GM₁

500 mg of di-lyso GM₁ (0.39 mM) (prepared according to Example 1) aredissolved in 5 ml of dimethylformamide, and to this are then slowlyadded 145 mg (0.43 mM) of9-fluorenylmethyl-oxycarbonyl-N-hydroxysuccinimide (FMOC-succ.). Themixture is left to react for 1 hr at room temperature.

At the end of the reaction, it is precipitated in 100 ml of acetone,filtered and dried. The product is then purified by silica gelchromatography using as elution solvent a mixture ofchloroform/methanol/water 60:30:6.

The fractions containing the intermediate reaction productN-FMOC-di-lyso-GM₁ are pooled, dried and then redissolved in 2.5 ml ofdimethylformamide/methanol 1:1 after which are added, at 0° C., 1.1 ml(7.92 mM) of triethylamine and 0.40 ml (3.96 mM) of methyltrifluoroacetate and it is reacted at room temperature for 3 days.

To this is then added 1 ml of piperidine to remove the fluorenyl group.It is left to react for 18 hrs at room temperature and precipitated in100 ml of acetone/water 9:1, filtered and dried.

The intermediate reaction product is dissolved in 2.5 ml ofdimethylformamide/methanol 1:1 after which are added, at roomtemperature, 1056 μl (7.6 mM) of triethylamine, 330 mg (1.9 mM) of2-pyridylacetic acid hydrochloride and 194.2 mg (0.76 mM) ofchloromethylpyridinium iodide dissolved in 2.5 ml of dimethylformamide.

This is left to react for 18 hrs at room temperature, precipitated in 50ml of acetone, dissolved with 5 ml of water and brought to pH 9.0 withNaOH 0.01N. This solution is left to react at room temperature for 2 hrsto remove the trifluoroacetyl group. It is dialyzed, concentrated to 3ml and precipitated in 15 ml of acetone.

The raw product obtained is purified by silica gel chromatography usingas eluent a mixture of chloroform/methanol/water 60:35:8.

The pure fractions are pooled, evaporated, redissolved in 2 ml ofchloroform/methanol 1:1 and precipitated in 10 ml of acetone.

Product obtained=296 mg (55% theoretical) Silica gel chromatography witha solvent formed by chloroform/methanol/CaCl₂ 0.3% 50:42:11, shows thecompound to be unitary with Rf=0.19. It is positive to ninhydrinstaining and shows a fluorometric reading of 254 nm.

EXAMPLE 20 N,N'-di-cycloheptanecarbonyl-di-lyso GM₁

500 mg (0.39 mM) of di-lyso GM₁ (prepared according to Example 1) aredissolved in 2.5 ml of dimethylformamide, after which are added at roomtemperature 0.33 ml (2.37 mM) of triethylamine, 162 μl (1.18 mM) ofcycloheptanecarboxy acid and 0.2 g (0.79 mM) of chloromethylpyridiniumiodide dissolved in 2.5 ml of dimethylformamide.

It is left to react for 18 hrs at room temperature and then precipitatedin 100 ml of acetone, filtered and dried. The product is then purifiedby silica gel chromatography using as eluent a mixture ofchloroform/methanol/water 60:25:4.

The pure fractions are pooled, evaporated, treated with Na₂ CO₃ 1N,dialyzed against distilled water and then concentrated to 5 ml andprecipitated in 50 ml of acetone.

Product obtained: 407 mg (68% theoretical).

Silica gel chromatography with a solvent formed bychloroform/methanol/CaCl₂ 0.3% 60:35:8 shows the compound to be unitarywith Rf=0.32.

EXAMPLE 21 N-N'-di-cyclopentanecarbonyl-di-lyso GM₁

500 mg (0.39 mM) of di-lyso GM₁ (prepared according to Example 1) aredissolved in 2.5 ml of dimethylformamide after which are added at roomtemperature 0.33 ml (2.37 mM) of triethylamine, 123 μl (1.18 mM) ofcyclopentanecarboxy acid and 0.2 g (0.79 mM) of chloromethylpyridiniumiodide dissolved in 2.5 ml of dimethylformamide.

The mixture is left to react for 18 hrs at room temperature and thenprecipitated in 100 ml of acetone. It is filtered and dried. The productis then purified by silica gel chromatography using as eluent a mixtureof chloroform/methanol/water 60:25:4.

The pure fractions are pooled, evaporated, treated with Nahd 2CO₃ 1N,dialyzed against distilled water and then concentrated to 5 ml andprecipitated in 50 ml of acetone.

Product obtained: 277 mg (48% theoretical).

Silica gel chromatography with a solvent formed bychloroform/methanol/CaCl₂ 0.3% 60:35:8 shows the compound to be unitarywith Rf=0.30.

EXAMPLE 22 N,N'-di-phenylacetyl-di-lyso GM₁

500 mg (0.39 mM) of di-lyso GM₁ (prepared according to Example 1) aredissolved in 2.5 ml of dimethylformamide after which are added at roomtemperature 0.88 ml (6.34 mM) of triethylamine, 430 mg (3.17 mM) ofphenylacetic acid and 0.2 g (0.79 mM) of chloromethylpyridinium iodidedissolved in 2.5 ml of dimethylformamide.

It is left to react for 18 hrs at room temperature and then precipitatedin 100 ml of acetone, filtered and dried. The product is then purifiedby silica gel chromatography using as eluent a mixture ofchloroform/methanol/water 60:25:4.

The pure fractions are pooled, evaporated, treated with Na₂ CO₃ 1N,dialyzed against distilled water and then concentrated to 5 ml andprecipitated in 50 ml of acetone.

Product obtained: 564 mg (95% theoretical).

Silica gel chromatography with a solvent formed bychloroform/methanol/CaCl₂ 0.3% 60:35:8 shows the compound to be unitarywith Rf=0.32. It shows a fluorometric reading of 254 nm.

EXAMPLE 23 N,N'-di-(5-methoxy-1-indanone-3-acetyl)-di-lyso GM₁

500 mg (0.39 mM) of di-lyso GM₁ (prepared according to Example 1) aredissolved in 2.5 ml of dimethylformamide after which are added at roomtemperature 0.88 ml (6.34 mM) of triethylamine, 700 mg (3.17 mM) of5-methoxy-1-indanone-3-acetic acid and 0.2 g (0.79 mM) ofchloromethylpyridinium iodide dissolved in 2.5 ml of dimethylformamide.

It is left to react for 18 hrs at room temperature and then precipitatedin 100 ml of acetone, filtered and dried. The product is then purifiedby silica gel chromatography using as eluent a mixture ofchloroform/methanol/water 60:25:4.

The pure fractions are pooled, evaporated, gathered with Na₂ CO₃ 1N,dialyzed against distilled water and then concentrated to 5 ml andprecipitated in 50 ml of acetone.

Product obtained: 601 mg (91% theoretical).

Silica gel chromatography with a solvent formed bychloroform/methanol/CaCl₂ 0.3% 60:35:8 shows the compound to be unitarywith Rf=0.33, displaying a fluorometric reading of 254 nm.

EXAMPLE 24 N,N'-di-(2-pyridylacetyl)-di-lyso GM₁

500 mg (0.39 mM) of di-lyso GM₁ (prepared according to Example 1) aredissolved in 2.5 ml of dimethylformamide after which are added at roomtemperature 0.88 ml (6.34 mM) of triethylamine, 550 mg (3.17 mM) of2-pyridylacetic acid and 0.2 g (0.79 mM) of chloromethylpyridiniumiodide dissolved in 2.5 ml of dimethylformamide.

It is left to react for 18 hrs at room temperature and then precipitatedin 100 ml of acetone, filtered and dried. The product is then purifiedby silica gel chromatography using as eluent a mixture ofchloroform/methanol/water 60:25:4.

The pure fractions are pooled, evaporated, treated with Na₂ CO₃ 1N,dialyzed against distilled water and then concentrated to 5 ml andprecipitated in 50 ml of acetone.

Product obtained: 268 mg (43% theoretical).

Silica gel chromatography with a solvent formed bychloroform/methanol/CaCl₂ 0.3% 60:35:8 shows the compound to be unitarywith Rf=0.23. It shows a fluorometric reading of 254 nm.

EXAMPLE 25 N,N'-di-(5-methyl-2-thiophenecarbonyl)-di-lyso GM₁

500 mg (0.39 mM) of di-lyso GM₁ (prepared according to Example 1) aredissolved in 2.5 ml of dimethylformamide after which are added at roomtemperature 0.88 ml (6.34 mM) of triethylamine, 450 mg (3.17 mM) of5-methyl-2-thiophene carboxy acid and 0.2 g (0.79 mM) ofchloromethylpyridinium iodide dissolved in 2.5 ml of dimethylformamide.

It is left to react for 18 hrs at room temperature and then precipitatedin 100 ml of acetone, filtered and dried. The product is then purifiedby silica gel chromatography using as eluent a mixture ofchloroform/methanol/water 60:25:4.

The pure fractions are pooled, evaporated, treated with Na₂ CO₃ 1N,dialyzed against distilled water and then concentrated to 5 ml andprecipitated in 50 ml of acetone.

Product obtained: 509 mg (85% theoretical).

Silica gel chromatography with a solvent formed bychloroform/methanol/CaCl₂ 0.3% 60:35:8 shows the compound to be unitarywith Rf=0.33. It shows a fluorometric reading of 254 nm.

EXAMPLE 26 N-acetyl-N'-2-pyridylacetyl-di-lyso GM₁

500 mg of di-lyso GM₁ (prepared according to Example 1) are dissolved in5 ml of dimethylformamide, and to this solution are then slowly added145 mg (0.43 mM) of 9-fluorenylmethyloxycarbonyl-N- -hydroxysuccinimide(FMOC-succ) and it is left to react for 1 hr at room temperature.

At the end of the reaction, it is precipitated in 100 ml. of acetone,filtered and dried. The product is then purified by silica gelchromatography using as elution solvent a mixture ofchloroform/methanol/water 60:30:6.

The fractions containing the intermediate reaction product(N-FMOC-di-lyso-GM₁) are pooled, dried and then redissolved in 2.5 ml ofdimethylformamide/methanol 1:1 after which are added, at roomtemperature, 1056 μl (7.6 mM) of triethylamine, 330 mg (1.9 mM) of2-pyridylacetic acid hydrochloride and 194.2 mg (0.76 mM) ofchloromethylpyridinium iodide dissolved in 2.5 ml of dimethylformamide.The mixture is reacted for 18 hrs at room temperature.

To this reaction mixture is then added 1 ml of piperidine to remove thefluorenyl group. It is left to react for 18 hrs at room temperature andprecipitated in 100 ml of acetone/water 9:1, filtered and dried.

The intermediate reaction product is dissolved in 30 ml ofchloroform/methanol/water 1:1:0.1 after which are added 1.1 ml (7.92 ml)of triethylamine and 373 μl (3.96 mM) of acetic anhydride. It is left toreact for 2 hrs at room temperature, dried, treated with 5 ml of Na₂ CO₃1M and kept at 60° C. for 1 hr. It is dialyzed, concentrated to 5 ml andprecipitated in 5 volumes of acetone.

The raw reaction product is purified by silica gel chromatography usingas elution solvent a mixture of chloroform/methanol/water 60:35:8.

The pure fractions are pooled, evaporated, redissolved in 2.0 ml ofchloroform/methanol 1:1 and precipitated in 10 ml of acetone.

Product obtained=299 mg (54% theoretical)

Silica gel chromatography with a solvent formed bychloroform/methanol/CaCl₂ 0.3% 50:42:11, shows the compound to beunitary with Rf=0.37. It shows a fluorometric reading of 254 nm.

EXAMPLE 27 N-acetyl-N'-3,4,5-trimethoxybenzoyl-di-lyso GM₁

500 mg of di-lyso GM₁ (0.39 mM) (prepared according to Example 1) aredissolved in 5 ml of dimethylformamide, and to this solution are thenslowly added 145 mg (0.43 mM) of9-fluorenylmethyloxy-carbonyl-N-hydroxysuccinimide (FMOC-succ). It isleft to react for 1 hr at room temperature. At the end of the reaction,it is precipitated in 100 ml of acetone, filtered and dried. The productis then purified by silica gel chromatography using as elution solvent amixture of chloroform/methanol/water 60:30:6. The fractions containingthe intermediate reaction product (N-FMOC-di-lyso-GM₁) are pooled, driedand then redissolved in 2.5 ml of dimethylformamide/methanol 1:1 and tothis solution is added trimethoxybenzoic anhydride dissolved in 20 ml oftetrahydrofuran, freshly prepared by reacting 0.3 g (1.9 mM) ofdicyclohexylcarbodiimide and 0.3 g (1.4 mM) of trimethoxybenzoic acid in20 ml of tetrahydrofuran and filtering away 2 hrs later thedicyclohexylurea which has formed.

The condensation reaction is conducted at 25° C. for 18 hrs understirring.

To this reaction mixture is then added 1 ml of piperidine to remove thefluorenyl group. It is left to react for 18 hrs at room temperature andprecipitated in 100 ml of acetone/water 9:1, filtered and dried.

The intermediate reaction product is dissolved in 30 ml ofchloroform/methanol/water 1:1:0.1 after which are added 1.1 ml (7.92 ml)of triethylamine and 373 μl (3.96 mM) of acetic anhydride. It is left toreact for 2 hrs at room temperature, dried, treated with 5 ml of Na₂ CO₃1M and kept at 60° C. for 1 hr. It is dialyzed, concentrated to 5 ml andprecipitated in 5 volumes of acetone.

The raw reaction product is purified by silica gel chromatography usingas elution solvent a mixture of chloroform/methanol/water 60:35:8.

The pure fractions are pooled, evaporated, redissolved in 2.0 ml ofchloroform/methanol 1:1 and precipitated in 10 ml of acetone.

Product obtained=286 mg (49% theoretical)

Silica gel chromatography with a solvent formed bychloroform/methanol/CaCl₂ 0.3% 50:42:11, shows the compound to beunitary with Rf=0.39. It shows a fluorometric reading of 254 nm.

EXAMPLE 28 N-dichloroacetyl-N'-2-pyridylacetyl-di-lyso GM₁

500 mg of di-lyso GM₁ (0.39 mM) (prepared according to Example 1) aredissolved in 5 ml of dimethylformamide, and to this solution are thenslowly added 145 mg (0.43 mM) of 9-fluorenylmethyloxy-carbonyl-N-hydroxysuccinimide (FMOC-succ). It is left to react for 1 hrat room temperature.

At the end of the reaction, it is precipitated in 100 ml of acetone,filtered and dried. The product is then purified by silica gelchromatography using as elution solvent a mixture ofchloroform/methanol/water 60:30:6. The fractions containing theintermediate reaction product (N-FMOC-di-lyso-GM₁) are pooled, dried andthen redissolved in 2.5 ml of dimethylformamide/methanol 1:1 after whichare added, at room temperature, 1056 μl (7.6 mM) of triethylamine, 330mg (1.9 mM) of 2-pyridylacetic acid hydrochloride and 194.2 g (0.76 mM)of chloromethylpyridinium iodide dissolved in 2.5 ml ofdimethylformamide. It is reacted at room temperature for 18 hr.

To this reaction mixture is then added 1 ml of piperidine to remove thefluorenyl group. It is left to react for 18 hrs at room temperature andprecipitated in 100 ml of acetone/water 9:1, filtered and dried.

The intermediate reaction product is dissolved in 30 ml ofchloroform/methanol/water 1:1:0.1 after which are added 1.1 ml (7.92 ml)of triethylamine and 950 mg (3.96 mM) of dichloroacetic anhydride. It isleft to react for 2 hrs at room temperature, dried, treated with 5 ml ofNa₂ CO₃ 1M and kept at 60° C. for 1 hr. It is dialyzed, concentrated to5 ml and precipitated in 5 volumes of acetone.

The reaction product is purified by silica gel chromatography using aselution solvent a mixture of chloroform/methanol/water 60:35:8.

The pure fractions are pooled, evaporated, redissolved in 2.0 ml ofchloroform/methanol 1:1 and precipitated in 10 ml of acetone.

Product obtained=267 mg (46% theoretical)

Silica gel chromatography with a solvent formed bychloroform/methanol/CaCl₂ 0.3% 50:42:11, shows the compound to beunitary with Rf=0.40. It shows a fluorometric reading of254 nm.

EXAMPLE 29 N-dichloroacetyl-N'-3,4,5-trimethoxybenzoyl-di-lyso GM₁

500 mg of di-lyso GM (0.39 mM) (prepared according to Example 1) aredissolved in 5 ml of dimethylformamide, and to this solution are thenslowly added 145 mg (0.43 mM) of 9-fluorenylmethyloxy-carbonyl-N-hydroxysuccinimide (FMOC-succ) and it is left to react for 1hr at room temperature.

At the end of the reaction, the resulting mixture is precipitated in 100ml of acetone, filtered and dried. The product is then purified bysilica gel chromatography using as elution solvent a mixture ofchloroform/methanol/water 60:30:6.

The fractions containing the intermediate reaction product(N-FMOC-di-lyso-GM₁) are pooled, dried and then redissolved in 2.5 ml ofdimethylformamide/methanol 1:1 and to this is added trimethoxybenzoicanhydride dissolved in 10 ml of tetrahydrofuran, freshly prepared byreacting 0.3 g (1.9 mM) of dicyclohexylcarbodiimide and 0.3 g (1.4 mM)of trimethoxybenzoic acid in 10 ml of tetrahydrofuran and filtering away2 hrs later the dicyclohexylurea which has formed.

The condensation reaction is conducted at 25° C. for 18 hrs understirring.

To this is then added 1 ml of piperidine to remove the fluorenyl group,and the mixture is left to react for 18 hrs at room temperature andprecipitated in 100 ml of acetone/water 9:1, filtered and dried. Theintermediate reaction product is dissolved in 30 ml ofchloroform/methanol/water 1:1:0.1 after which are added 1.1 ml (7.92 ml)of triethylamine and 950 mg (3.96 mM) of dichloroacetic anhydride. It isleft to react for 2 hrs at room temperature, dried, treated with 5 ml ofNa₂ CO₃ 1M and kept at 60° C. for 1 hr. It is dialyzed, concentrated to5 ml and precipitated in 5 volumes of acetone.

The raw reaction product is purified by silica gel chromatography usingas elution solvent a mixture of chloroform/methanol/water 60:35:8.

The pure fractions are pooled, evaporated, redissolved in 2.0 ml ofchloroform/methanol 1:1 and precipitated in 10 ml of acetone.

Product obtained=293 mg (48% theoretical)

Silica gel chromatography with a solvent formed bychloroform/methanol/CaCl₂ 0.3% 50:42:11, shows the compound to beunitary with Rf=0.41 and having a fluorometric reading of 254 nm.

EXAMPLE 30 N-2-furoyl-N'-butyryl-di-lyso GM₁

500 mg of di-lyso GM₁ (0.39 mM) (prepared according to Example 1) aredissolved in 5 ml of dimethylformamide, and to this solution are thenslowly added 145 mg (0.43 mM) of9-fluorenylmethyl-oxycarbonyl-N-hydroxysuccinimide (FMOC-succ). It isleft to react for 1 hr at room temperature.

At the end of the reaction, it is precipitated in 100 ml of acetone,filtered and dried. The product is then purified by silica gelchromatography using as elution solvent a mixture ofchloroform/methanol/water 60:30:6. The fractions containing theintermediate reaction product (N-FMOC-di-lyso-GM₁) are pooled, dried andthen redissolved in 2.5 ml of dimethylformamide/methanol 1:1 after whichare added, at 0° C., 1.1 ml (7.92 mM) of triethylamine and 626 mg (3.96mM) of butyric anhydride. It is left to react at room temperature for 2hrs.

To this reaction mixture is then added 1 ml of piperidine to remove thefluorenyl group, and it is left to react for 18 hrs at room temperatureand precipitated in 100 ml of acetone/water 9:1, filtered and dried.

The intermediate reaction product is dissolved in 2.5 ml ofdimethylformamide after which are added at room temperature 528 μl (3.8mM) of triethylamine, 210 mg (1.9 mM) of 2-furoic acid and 194.2 mg(0.76 Mm) of chloromethylpyridinium iodide dissolved in 2.5 ml ofdimethylformamide.

It is left to react for 18 hrs at room temperature and then precipitatedin 100 ml of acetone, filtered and dried. The product is then purifiedby silica gel chromatography using as eluent a mixture ofchloroform/methanol/water 60:35:8.

The pure fractions are pooled, evaporated, redissolved in 2.0 ml ofchloroform/methanol 1:1 and precipitated in 10 ml of acetone.

Product obtained=289 mg (52% theoretical)

Silica gel chromatography with a solvent formed bychloroform/methanol/CaCl₂ 0.3% 50:42:11, shows the compound to beunitary with Rf=0.39. It shows a fluorometric reading of 254 nm.

EXAMPLE 31 N-2,6-dimethoxybenzoyl-N'-butyryl-di-lyso GM₁

500 mg of di-lyso GM₁ (0.39 Mm) (prepared according to Example 1) aredissolved in 5 ml of dimethylformamide, and to this solution are thenslowly added 145 mg (0.43 mM) of 9-fluorenylmethyloxy-carbonyl-N-hydroxysuccinimide (FMOC-succ). It is left to react for 1 hrat room temperature.

At the end of the reaction, it is precipitated in 100 ml of acetone,filtered and dried. The product is then purified by silica gelchromatography using as elution solvent a mixture ofchloroform/methanol/water 60:30:6. The fractions containing theintermediate reaction product (N-FMOC-di-lyso-GM₁) are pooled, dried andthen redissolved in 2.5 ml of dimethylformamide/methanol 1:1 after whichare added, at 0° C., 1.1 ml (7.92 mM) of triethylamine and 626 mg (3.96mM) of butyric anhydride and it is reacted at room temperature for 2hrs.

To this reaction mixture is then added 1 ml of piperidine to remove thefluorenyl group. It is left to react for 18 hrs at room temperature andprecipitated in 100 ml of acetone/water 9:1, filtered and dried.

The intermediate reaction product is dissolved in 1 ml ofdimethylformamide after which are added at room temperature 1.056 ml(7.6 mM) of triethylamine and 2,6-dimethoxybenzoic anhydride, freshlyprepared by reacting 2.76 g (15.2 mM) of 2,6-dimethoxybenzoic acid and1.08 g (3.8 mM) of fluoromethylpyridinium para-toluenesulfonate in 10 mlof dimethylformamide/tetrahydrofuran 1:1. The condensation reaction isconducted at room temperature for 4 hrs under stirring. At the end ofthe reaction, the solution is concentrated to 5 ml, precipitated in 50ml of acetone, filtered and vacuum-dried.

Silica gel chromatography is effected, using as eluent a mixture ofchloroform/methanol/water 60:35:8.

The pure fractions are pooled, evaporated, treated with Na₂ CO₃ 1N,dialyzed against distilled water and then concentrated to 5 ml andprecipitated in 50 ml of acetone.

Product obtained=297 mg (51% theoretical)

Silica gel chromatography with a solvent formed bychloroform/methanol/CaCl₂ 0.3% 50:42:11, shows the compound to beunitary with Rf=0.37. It shows a fluorometric reading of 254 nm.

EXAMPLE 32 N-2-pyridylacetyl-N'-lauroyl-di-lyso GM₁

500 mg of di-lyso GM₁ (0.39 mM) (prepared according to Example 1) aredissolved in 5 ml of dimethylformamide, and to this solution are thenslowly added 145 mg (0.43 mM) of 9-fluorenylmethyloxy-carbonyl-N-hydroxysuccinimide (FMOC-succ). It is left to react for 1 hrat room temperature.

At the end of the reaction, it is precipitated in 100 ml of acetone,filtered and dried. The product is then purified by silica gelchromatography using as elution solvent a mixture ofchloroform/methanol/water 60:30:6.

The fractions containing the intermediate reaction product(N-FMOC-di-lyso-GM₁) are pooled, dried and then redissolved in 2.5 ml ofdimethylformamide/methanol 1:1 after which are added, at 0° C., 1.1 ml(7.92 mM) of triethylamine and 1.51 g (3.96 mM) of lauric anhydride andthis is reacted at room temperature for 18 hrs.

To this reaction mixture is then added 1 ml of piperidine to remove thefluorenyl group. It is left to react for 18 hrs at room temperature andprecipitated in 100 ml of acetone/water 9:1, filtered and dried.

The intermediate reaction product is dissolved in 2.5 ml ofdimethylformamide/methanol 1:1 after which are added at room temperature1056 μl (7.6 ml) of triethylamine, 330 mg (1.9 mM) of 2-pyridylaceticacid hydrochloride and 194.2 mg (0.76 mM) of chloromethylpyridiniumiodide dissolved in 2.5 ml of dimethylformamide. It is reacted for 18hrs at room temperature and then precipitated in 100 ml of acetone.

The raw reaction product is purified by silica gel chromatography usingas elution solvent a mixture of chloroform/methanol 60:35:8.

The pure fractions are pooled, evaporated, redissolved in 2.0 ml ofchloroform/methanol 1:1 and precipitated in 10 ml of acetone.

Product obtained=262 mg (43% theoretical)

Silica gel chromatography with a solvent formed bychloroform/methanol/CaCl₂ 0.3% 50:42:11, shows the compound to beunitary with Rf=0.61. It displays a fluorometric reading of 254 nm.

EXAMPLE 33 N'-3,4,5-trimethoxybenzoyl-N'-lauroyl-di-lyso GM₁

500 mg of lyso GM₁ (0.39 mM) (prepared according to Example 1) aredissolved in 5 ml of dimethylformamide, and to this are then slowlyadded 145 mg (0.43 mM) of9-fluorenylmethyloxycarbonyl-N-hydroxysuccinimide (FMOC-succ). It isleft to react for 1 hr at room temperature. At the end of the reactionit is precipitated in 100 ml of acetone, filtered and dried. The productis then purified by silica gel chromatography using as elution solvent amixture of chloroform/methanol/water 60:30:6.

The fractions containing the intermediate reaction product(N-FMOC-di-lyso-GM₁) are pooled, dried and then redissolved in 2.5 ml ofdimethyl formamide/methanol 1:1 after which are added, at 0° C., 1.1 ml(7.92 mM) of triethylamine and 1.51 g (3.96 mM) of lauric anhydride. Itis reacted at room temperature for 18 hrs.

To this reaction mixture is then added 1 ml of piperidine to remove thefluorenyl group. It is left to react for 18 hrs at room temperature andprecipitated in 100 ml of acetone/water 9:1, filtered and dried.

The intermediate reaction product is dissolved in 2.5 ml ofdimethylformamide/methanol 1:1 and to this solution is addedtrimethoxybenzoic anhydride dissolved in 10 ml of tetrahydrofuran,freshly prepared by reacting 0.3 g (1.9 Mm) of dicyclohexylcarbodiimideand 0.3 g (1.4 mM) of trimethoxybenzoic acid in 10 ml of tetrahydrofuranand filtering away, 2 hrs later, the dicyclohexylurea which has formed.

The condensation reaction is conducted at 25° C. for 18 hrs understirring and then the product is precipitated in 100 ml of acetone.

The raw reaction product is purified by silica gel chromatography usingas elution solvent a mixture of chloroform/methanol/water 60:35:8.

The pure fractions are pooled, evaporated, redissolved in 2.0 ml ofchloroform/methanol 1:1 and precipitated in 10 ml of acetone.

Product obtained=294 mg (46% theoretical)

Silica gel chromatography with a solvent formed bychloroform/methanol/CaCl₂ 0.3% 50:42:11 shows the product to be aunitary compound with Rf=0.63. Its fluorometric reading is 254 nm.

EXAMPLE 34 N-3,4,5-trimethoxybenzoyl-N'-2-pyridylacetyl-di-lyso GM₁

500 mg of di-lyso GM₁ (0.39 mM) (prepared according to example 1) aredissolved in 5 ml of dimethylformamide.

To this solution are then slowly added 145 mg (0.43 mM) of9-fluorenylmethyloxycarbonyl-N-hydroxysuccinimide (FMOC-succ) and it isleft to react for 1 hour at room temperature.

At the end of the reaction, it is precipitated in 100 ml of acetone,filtered and dried. The product is then purified by silica gelchromatography using as elution solvent a mixture ofchloroform/methanol/water 60:30:6.

The fractions containing the intermediate reaction product(N-FMOC-di-lyso-GM₁) are pooled, dried and then redissolved in 2.5 ml ofdimethylformamide/methanol 1:1 and to this solution are added, at roomtemperature, 1056 μl (7.6 mM) of triethylamine and 330 mg (1.9 mM) of 2-pyridylacetic acid hydrochloride and 194.2 mg (0.76 mM) ofchloromethylpyridinium iodide dissolved in 2.5 ml of dimethylformamide.It is left to react at room temperature for 18 hrs.

To this reaction mixture is then added 1 ml of piperidine to remove thefluorenyl group. It is left to react for 18 hrs at room temperature andprecipitated in 100 ml of acetone/water 9:1, filtered and dried.

The intermediate reaction product is dissolved in 2.5 ml ofdimethylformamide/methanol 1:1 and to this solution is addedtrimethoxybenzoic anhydride dissolved in 10 ml of tetrahydrofuran,freshly prepared by reacting 0.3 g (1.9 mM) of dicyclohexylcarbodiimideand 0.3 g (1.4 mM) of trimethoxybenzoic acid in 10 ml of tetrahydrofuranand filtering away, 2 hrs later, the dicyclohexylurea which has formed.

The condensation reaction is conducted at 25° C. for 18 hrs understirring and then the product is precipitated in 100 ml of acetone.

The raw reaction product is purified by silica gel chromatography usingas elution solvent a mixture of chloroform/methanol/water 60:35:8.

The pure fractions are pooled, evaporated, redissolved in 2.0 ml ofchloroform/methanol 1:1 and precipitated in 10 ml of acetone.

Product obtained=243 mg (43% theoretical)

Silica gel chromatography with a solvent formed bychloroform/methanol/CaCl₂ 0.3% 50:42:11 shows the product to be aunitary compound with Rf=0.59. Its fluorometric reading is 254 nm.

EXAMPLE 35 N-2-pyridylacetyl-N'-2,6-dimethoxybenzoyl-di-lyso GM₁

500 mg of di-lyso GM₁ (0.39 mM) (prepared according to Example 1) aredissolved in 5 ml of dimethylformamide, and to this solution are thenslowly added 145 mg (0.43 mM) of9-fluorenylmethyloxycarbonyl-N-hydroxysuccinimide (FMOC-succ). It isleft to react for 1 hour at room temperature.

At the end of the reaction, it is precipitated in 100 ml of acetone,filtered and dried. The product is then purified by silica gelchromatography using as elution solvent a mixture ofchloroform/methanol/water 60:30:6.

The fractions containing the intermediate reaction product(N-FMOC-di-lyso-GM₁) are pooled, dried and then redissolved in 2.5 ml ofdimethylformamide/methanol 1:1 and to this solution is addeddimethoxybenzoic anhydride, freshly prepared by reacting 1.88 g (1.6 mM)of 2,6-dimethoxybenzoic acid and 540 mg (1.9 mM) offluoromethylpyridinium para-toluenesulfonate in 5 ml ofdimethylformamide/tetrahydrofuran 1:1, which is then left to react atroom temperature for 4 hrs.

1 ml of piperidine is then added to remove the fluorenyl groups. Themixture is left to react for 18 hrs at room temperature and thenprecipitated in 100 ml of acetone/water 9:1, filtered and dried.

The intermediate reaction product is dissolved in 2.5 ml ofdimethylformamide/methanol 1:1 and to this are added, at roomtemperature, 1056 μl (7.6 mM) of triethylamine, 330 mg (1.9 mM) of2-pyridylacetic acid hydrochloride and 194.2 mg (0.76 mM) ofchloromethylpyridinium iodide dissolved in 2.5 ml of dimethylformamide.It is reacted at room temperature for 18 hrs and then the product isprecipitated in 100 ml of acetone.

The raw reaction product is purified by silica gel chromatography usingas elution solvent a mixture of chloroform/methanol/water 60:35:8.

The pure fractions are pooled, evaporated, redissolved in 2.0 ml ofchloroform/methanol 1:1 and precipitated in 10 ml of acetone.

Product obtained=247 mg (41% theoretical)

Silica gel chromatography with a solvent formed bychloroform/methanol/CaCl₂ 0.3% 50:42:11 shows the product to be aunitary compound with Rf=0.58 with a fluorometric reading of 254 nm.

EXAMPLE 36 N'-3,4,5-trimethoxybenzoyl-N'-lyso GM₁

500 mg (0.33 mM) of N'-lyso GM₁ are dissolved in 50 ml ofchloroform/methanol 1:1 and to this solution is added 0.28 g (0.7 mM) oftrimethoxybenzoic anhydride dissolved in 20 ml of tetrahydrofuran, andfreshly prepared by reacting 0.3 g (1.9 mM) of dicyclohexylcarbodiimideand 0.3 g (1.4 mM) of trimethoxybenzoic acid in 20 ml oftetrahydrofuran, and filtering away, 2 hrs later, the dicyclohexylureawhich has formed.

The condensation reaction is conducted at 25° C. for 18 hrs understirring.

At the end of the reaction, the product is dried, gathered with 5 ml ofchloroform/methanol 1:1 and precipitated in100 ml of acetone.

The raw product thus obtained is purified by silica gel chromatographyusing as elution solvent a mixture of chloroform/methanol/water 60:15:2.

The pure fractions are pooled, evaporated, redissolved in 5 ml ofchloroform/methanol 1:1 and the product is precipitated with 100 ml ofacetone.

Product obtained: 320 mg (56.7% theoretical).

Silica gel chromatography with a solvent formed bychloroform/methanol/CaCl₂ 2 0.3% 60:35:8 shows the product to be afluorescent unitary compound with an Rf of 0.50.

EXAMPLE 37 N'-2-furoyl-di-lyso GM₁

500 mg of di-lyso GM₁ (0.39 mM) (prepared according to Example 1) aredissolved in 5 ml of dimethylformamide, and to this solution are thenslowly added 145 mg (0.43 mM) of9-fluorenylmethyloxycarbonyl-N-hydroxysuccinimide dissolved in 2 ml oftetrahydrofuran. It is left to react for 1 hour at room temperature.

At the end of the reaction, 528 μl (3.8 mM) of triethylamine, 210 mg(1.9 mM) of 2-furoic acid and 194.2 mg (0.76 mM) ofchloromethylpyridinium iodide dissolved in 2.5 ml of dimethylformamideare added at room temperature. This mixture is then left to react for 18hrs at room temperature after which are added 2 ml of piperidine toremove the protector group.

This is left to react for 18 hrs at room temperature and thenprecipitated in 100 ml of acetone, filtered and dried. The product thusobtained is dissolved in 10 ml Na₂ CO₃ 1M and kept at 60° C. for 1 hour.It is dialyzed, concentrated to 5 ml and precipitated in 5 volumes ofacetone.

The raw reaction product is purified by silica gel chromatography usingas elution solvent a mixture of chloroform/methanol/NH₃ 2.5N 60:35:8.

The fractions containing the pure product are dried and then redissolvedin 5 ml of water. The solution is brought to pH 10 with NaOH 0.01N anddialyzed, concentrated to 5 ml and precipitated in 50 ml of acetone.

Product obtained: 301 mg (57% theoretical).

Silica gel chromatography with a solvent formed bychloroform/methanol/CaCl₂ 0.3% 50:42:11 shows the product to be unitarywith Rf=0.31, positive to ninhydrin staining. Its fluorometric readingis 254 nm.

EXAMPLE 38 N'-4,5-trimethoxybenzoyl-di-lyso GM₁

500 mg of di-lyso GM₁ (0.39 mM) (prepared according to Example 1) aredissolved in 5 ml of dimethylformamide, and to this solution are thenslowly added 145 mg (0.43 mM) of9-fluorenylmethyloxycarbonyl-N-hydroxysuccinimide dissolved in 2 ml oftetrahydrofuran, and it is left to react for 1 hour at room temperature.

At the end of the reaction, trimethoxybenzoic anhydride is added,dissolved in 20 ml of tetrahydrofuran, freshly prepared by reacting 0.3g (1.9 mM) of dicyclohexylcarbodiimide and 0.3 g (1.4 mM) oftrimethoxybenzoic acid in 20 ml of tetrahydrofuran, and filtering away,2 hrs later, the dicyclohexylurea which has formed.

The condensation reaction is conducted at 25° C. for 18 hrs understirring. 2 ml of piperidine are then added to remove the protectorgroup, and it is left to react for 18 hrs at room temperature and thenprecipitated in 100 ml of acetone. It is filtered and dried. The productthus obtained is dissolved in 10 ml Na₂ CO₃ 1M and kept at 60° C. for 1hour. It is dialyzed, concentrated to 5 ml and precipitated in 5 volumesof acetone. The raw reaction product is purified by silica gelchromatography using as elution solvent a mixture ofchloroform/methanol/NH₃ 2.5N 60:35:8. The fractions containing the pureproduct are dried and then redissolved in 5 ml of water. It is broughtto pH 10 with NaOH 0.01N and dialyzed, concentrated to 5 ml andprecipitated in50 ml of acetone.

Product obtained: 317.5 mg (56% theoretical).

Silica gel chromatography with a solvent formed bychloroform/methanol/CaCl₂ 0.3% 50:42:11 shows the product to be unitarywith Rf=0.35, positive to ninhydrin staining. Its fluorometric readingis 254 nm.

EXAMPLE 39 Preparation of a Ganglioside Mixture (GA Mixture) byExtraction from Bovine Brain Tissue

Bovine brain cortex is removed from the animal and homogenized inphosphate buffer to pH 6.8; to this are then added 6 volumes oftetrahydrofuran and the resulting mixture is centrifuged. Thesupernatant is then reextracted twice with tetrahydrofuran. Aftercentrifugation the non-polar materials are removed by partitioning withethyl ether and the aqueous-tetrahydrofuran phase is introduced into theion exchange column equilibrated with 50% ethanol. To the product fromthe column is added barium hydroxide and four volumes of iced ethanol.After 18 hrs cooling, a precipitate is gathered which is then slightlyacidified with hydrochloric acid, after dissolution in water. Thesolution thus obtained is dialyzed and freeze-dried. The yield at thispoint is approx. 0.6 mg of raw ganglioside mixture per gram of nervoustissue. The freeze-dried powder is dispersed in 20 volumes ofchloroform-methanol 2:1, and once the solution obtained has beenfiltered to perfect clearness, it is then partitioned by adding 0.2volumes of a solution of potassium chloride in water at 0.88%.

The upper layer is separated, dialyzed and freeze-dried. The final yieldis approximately 0.3 mg of purified mixture of ganglioside salts pergram of brain tissue. The ganglioside mixture obtained can be fractionedin various portions representing substantially pure gangliosides (in thesense as already described above), using columns of silicic acid andeluting with mixtures of methanol-chloroform. Thus, an averagecomposition of approximately 40% of ganglioside GD_(1a), 21% ofganglioside GM₁, 19% of ganglioside GT_(1b) and 16% of gangliosideGD_(1b) is obtained.

EXAMPLE 40 2-furoyl derivatives of a mixture of N-lysogangliosides 1)Preparation of N-lysogangliosides

10 g of the ganglioside mixture (obtained according to Example 39 aredissolved in 200 ml of KOH 3N and hydrolysis is conducted for 72 hrs at90° C. The solution is then cooled and brought to pH 6.5 withhydrochloric acid. It is left to stand for 18 hrs at 4° C. and then theprecipitated fatty acids are filtered away. It is dialyzed against waterand concentrated to 500 ml and precipitated in 5 lt of acetone.

The product containing the N'-lysogangliosides andN,N'-di-lysogangliosides (<20%) is vacuum-dried and then redissolved in100 ml of dimethylformamide.

To this solution are then slowly added 2.15 g (6.37 mM) of9-fluorenylmethyloxycarbonyl-N-hydroxysuccinimide dissolved in 20 ml oftetrahydrofuran and it is left to react for 1 hour at room temperature.Finally, 3 ml (31.85 mM) of acetic anhydride and 0.9 ml (63.7 mM) oftriethylamine are added. After 30 minutes, 12.5 ml of piperidine areadded to remove the protecting group. It is left to react for 18 hrs atroom temperature and precipitated in 2 lt of acetone and dried. Thematerial thus obtained is dissolved in Na₂ CO₃ 1M and kept at 60° for 1hour. It is dialyzed, concentrated to 100 mg/ml and precipitated in 5volumes of acetone. The product is passed through an S-Sepharose column(H⁺ form) equilibrated in methanol. It is washed with methanol, thusobtaining the N-lysogangliosides by eluting with NH₄ Cl 10 mM inmethanol.

The fractions containing the product are dried and then redissolved inwater. They are brought to pH 10 with NaOH 0.01N and dialyzed,concentrated to 100 mg/ml and precipitated in 5 volumes of acetone.

Product obtained: 4.7 g (55% theoretical).

2) Preparation of the 2-furoyl Derivative

500 mg (0.31 mM) of the previously prepared mixture ofN-lysogangliosides are dissolved in 2.5 ml of dimethylformamide and tothis solution are added 431 μl (3.1 mM) of triethylamine, 174 mg (1.55mM) of 2-furoic acid and 158.4 mg (0.62 mM) of1-methyl-2-chloropyridinium iodide dissolved in 2.5 ml ofdimethylformamide.

It is reacted for 18 hrs at room temperature and then the product isprecipitated in 10 ml of acetone. It is filtered and dried.

The acylated product is separated from the compound which has notreacted by chromatography on an S-Sepharose column (H⁺ form)equilibrated in methanol. The furoyl derivative is eluted in methanol,dried, gathered with Na₂ CO₃ 1N, dialyzed, concentrated to 2.5 ml andprecipitated in 25 ml of acetone.

Product obtained: 373 mg (72% theoretical).

EXAMPLE 41 Methyl ester of N-(2-furoyl)-N-lyso GM₁

500 mg (0.36 mM) of the N-(2-furoyl)-N-lyso GM₁ sodium salt (preparedaccording to Example 7) are dissolved in 5 ml of N-methylpyrrolidone andto this solution are added 44.5 μl (0.72 mM) of methyl iodide. It isleft to react for 3 hrs at room temperature, precipitated in ethylacetate, filtered and vacuum-dried.

The product is then purified by silica gel chromatography using aseluent a mixture of chloroform/methanol/water (60:30:6).

The pure fractions are pooled, evaporated, redissolved in 2.5 ml ofchloroform/methanol 1:1 and precipitated in 25 ml of acetone.

Product obtained: 449 mg (89% theoretical).

Silica gel chromatography with a solvent formed bychloroform/methanol/CaCl₂ 0.3% (50:42:11), shows the compound to beunitary with Rf=0.4 [N-(2-furoyl) lyso GM₁ =0.37].

EXAMPLE 42 Peracetylate of the methyl ester of N-(2-furoyl)-N-lyso GM₁

500 mg (0.36 mM) of the methyl ester of N-(2-furoyl)-N-lyso GM₁(prepared according to Example 41) are dissolved in 5 ml of pyridine andto this solution are added 2.5 ml of acetic anhydride, freshlydistilled, and the mixture is stirred for 72 hrs at room temperature. Atthe end of the reaction, the solution is evaporated in a rotaryevaporator and the residue is partitioned between 10 ml of iced waterand 10 ml of ethyl acetate; the ethyl acetate is washed in cold HCl 1M,with water and with a solution of NaHCO₃ 1M. The organic phases areanhydrified with sodium sulfate, evaporated and the residue is purifiedby silica gel chromatography, using a mixture of dichloromethane/ethylacetate/isopropanol (70:30:45). The pure fractions are pooled,evaporated, redissolved in 5 ml of ethyl ether and precipitated in 25 mlof n-hexane.

Product obtained: 463 mg (62% theoretical).

Silica gel chromatography with a solvent formed bychloroform/methanol/ethyl acetate (70:10:30) and ethylacetate/isopropanol (95:5), shows the product to be unitary with Rf of0.45 and 0.26, respectively.

EXAMPLE 43 Inner ester of N-(2-furoyl)-N-lyso GM₁

500 mg (0.36 mM) of N-(2-furoyl)-N-lyso GM₁ sodium salt are dissolved in5 ml of N-methylpyrrolidone at 4° C. and reacted with 55 μl (0.4 mM) oftriethylamine and 100 mg (0.41 mM) of 1-methyl-2-chloropyridiniumiodide. The reaction is conducted for 4 hrs with a quantitative yield.The product is precipitated by adding 50 ml of acetone, and it isfiltered, gathered with 5 ml of chloroform/isopropanol 1:1 andreprecipitated in 25 ml of acetone.

Product obtained: 476 mg (96% theoretical).

Silica gel chromatography with a solvent formed bychloroform/methanol/CaCl₂ 0.3% (50:42:11), shows the compound to beunitary with Rf=0.44 [N-(2-furoyl)-N-lyso GM₁ =0.37].

EXAMPLE 44 2-butylamide of N-(2-furoyl)-N-lyso GM₁

500 mg (0.36 mM) of the methyl ester of N-(2-furoyl)-N-lyso GM₁(prepared according to Example 41) are dissolved in 5 ml of pyridine andto this solution are added 2.5 ml of 2-butylamine. It is reacted for 72hrs at room temperature and then dried in a rotary evaporator, dissolvedwith 5 ml of chloroform/methanol 1:1 and precipitated in 25 ml ofacetone. It is filtered and vacuum-dried.

The product is then purified by silica gel chromatography using aseluent a mixture of chloroform/methanol/water (60:25:4).

The pure fractions are pooled, evaporated, redissolved in 2.5 ml ofchloroform/methanol 1:1 and precipitated in 25 ml of acetone.

Product obtained: 376 mg (75% theoretical).

Silica gel chromatography with a solvent formed bychloroform/methanol/CaCl₂ 0.3% (50:42:11), shows the compound to beunitary with Rf=0.50 [methyl ester of N-(2-furoyl)-N-lyso GM₁ =0.42].

Pharmaceutical Preparations in Injectable Solutions EXAMPLE 45

    ______________________________________                                        Preparation No. 1 - one 2 ml vial contains:                                   ______________________________________                                        active substance         5 mg                                                 sodium chloride         16 mg                                                 citrate buffer pH 6 in   2 ml                                                 water for injection to                                                        ______________________________________                                    

The active substance is chosen from the group formed by the gangliosidederivatives described in either one of Examples 5 and 12.

    ______________________________________                                        Preparation No. 2 - one 2 ml vial contains:                                   ______________________________________                                        active substance        50 mg                                                 sodium chloride         16 mg                                                 citrate buffer pH 6 in   2 ml                                                 water for injection to                                                        ______________________________________                                    

The active substance is chosen from the group formed by the gangliosidederivatives described in Example 7.

    ______________________________________                                        Preparation No. 3 - one 4 ml flacon contains:                                 ______________________________________                                        active substance        100 mg                                                sodium chloride          32 mg                                                citrate buffer pH 6 in   4 ml                                                 water for injection to                                                        ______________________________________                                    

The active substance is chosen from the group formed by the gangliosidederivatives described in Examples 27, 29, 38 and 40.

Pharmaceutical Compositions Prepared in Twin Flacons EXAMPLE 46

The preparations illustrated in this Example are presented in twinflacons. The first flacon contains the active substance in the form of afreeze-dried powder in quantities varying between 10% and 90% by weighttogether with a pharmaceutically acceptable excipient, such as glycineor mannitol. The second flacon contains the solvent, as a sodiumchloride solution, and a citrate buffer. Immediately prior toadministration the contents of the two flacons are mixed together andthe freeze-dried powder containing the active substance dissolvesrapidly, forming an injectable solution. The pharmaceutical formcomprised of a flacon containing the active substance in the form of afreeze-dried powder, is the preferred form of the present invention.

    ______________________________________                                        System No. 1                                                                  ______________________________________                                        a. one 2 ml flacon of freeze-dried substance contains:                        ______________________________________                                        active substance           5 mg                                               glycine                   30 mg                                               b. one 2 ml vial of solvent contains:                                         ______________________________________                                        sodium chloride           16 mg                                               citrate buffer in water for injection to                                                                 2 ml                                               ______________________________________                                    

The active substance is chosen from the group formed by the gangliosidederivatives described in either one of Examples 5 and 12.

    ______________________________________                                        System No. 2                                                                  ______________________________________                                        a. one 3 ml vial of freeze-dried substance contains:                          ______________________________________                                        active substance           5 mg                                               mannitol                  40 mg                                               b. one 2 ml vial of solvent contains:                                         ______________________________________                                        sodium chloride           16 mg                                               citrate buffer in water for injection to                                                                 2 ml                                               ______________________________________                                    

The active substance is chosen from the group formed by the gangliosidederivatives described in either one of Examples 5 and 12.

    ______________________________________                                        System No. 3                                                                  ______________________________________                                        a. one 3 ml vial of freeze-dried substance contains:                          ______________________________________                                        active substance          50 mg                                               glycine                   25 mg                                               b. one 3 ml vial of solvent contains:                                         ______________________________________                                        sodium chloride           24 mg                                               citrate buffer in water for injection to                                                                 3 ml                                               ______________________________________                                    

The active substance is chosen from the group formed by the gangliosidederivatives described in any one of Examples 18, 33 and 38.

    ______________________________________                                        System No. 4                                                                  ______________________________________                                        a. one 3 ml vial of freeze-dried substance contains:                          ______________________________________                                        active substance          50 mg                                               mannitol                  20 mg                                               b. one 3 ml vial of sobvent contains:                                         ______________________________________                                        sodium chloride           24 mg                                               citrate buffer in water for injection to                                                                 3 ml                                               ______________________________________                                    

The active substance is chosen from the group formed by the gangliosidederivatives described in any one of Examples 18, 33 and 38.

    ______________________________________                                        System No. 5                                                                  ______________________________________                                        a. one 5 ml flacon of freeze-dried substance contains:                        ______________________________________                                        active substance          150 mg                                              glycine                    50 mg                                              b. one 4 ml vial of solvent contains:                                         ______________________________________                                        sodium chloride            32 mg                                              citrate buffer in water for injection to                                                                 4 ml                                               ______________________________________                                    

The active substance is chosen from the group formed by the gangliosidederivatives described in any one of Examples 27, 29, 38 and 40.

    ______________________________________                                        System No. 6                                                                  ______________________________________                                        a. one 5 ml flacon of freeze-dried substance contains:                        ______________________________________                                        active substance          100 mg                                              mannitol                   40 mg                                              b. one 4 ml vial of solvent contains:                                         ______________________________________                                        sodium chloride            32 mg                                              citrate buffer in water for injection to                                                                 4 ml                                               ______________________________________                                    

The active substance is chosen from the group formed by the gangliosidederivatives described in any one of Examples 27, 29, 38 and 40.

    ______________________________________                                        System No. 7                                                                  ______________________________________                                        a. one 3 ml flacon contains:                                                  ______________________________________                                        sterile, micronized active substance                                                                  40 mg                                                 b. one 3 ml vial of solvent contains:                                         ______________________________________                                        Tween 80                10 mg                                                 sodium chloride         24 mg                                                 phosphate buffer in      3 ml                                                 water for injection to                                                        ______________________________________                                    

The active substance is chosen from the group formed by the gangliosidederivatives described in any one of Examples 41, 42 and 43.

    ______________________________________                                        System No. 8                                                                  ______________________________________                                        a. one 5 ml flacon contains:                                                  ______________________________________                                        sterile, micronized active substance                                                                    100 mg                                              b. one 4 ml vial of solvent contains:                                         ______________________________________                                        Tween 80                   15 mg                                              soybean lecithin           5 mg                                               sodium chloride            36 mg                                              citrate buffer in water for injection to                                                                 4 ml                                               ______________________________________                                    

The active substance is chosen from the group formed by the gangliosidederivatives described in any one of Examples 41, 42 and 43.

Pharmaceutical Preparations for Transdermal Administration EXAMPLE 47

    ______________________________________                                        Preparation No. 1 - one plaster contains:                                     ______________________________________                                        active substance        100 mg                                                glycerol                 1.6 g                                                polyvinyl alcohol       200 mg                                                polyvinylpyrrolidone    100 mg                                                excipient to enhance                                                          transdermal penetration  20 mg                                                water                    1.5 g                                                ______________________________________                                    

The active substance is chosen from the group formed by the gangliosidederivatives described in any one of Examples 22, 23 and 25.

    ______________________________________                                        Preparation No. 2 - 100 g of ointment contain:                                ______________________________________                                        active substance (in 5 g of mixed                                                                       4.0 g                                               phospholipid liposomes)                                                       polyethylene glycol monostearate                                                                        1.5 g                                               glycerol                  1.5 g                                               ester of p-hydroxybenzoic acid                                                                          125 mg                                              water                    72.9 g                                               ______________________________________                                    

The active substance is chosen from the group formed by the gangliosidederivatives described in any one of Examples 22, 23 and 25.

Pharmaceutical Preparations for Oral Administration EXAMPLE 48

    ______________________________________                                        Preparation No. 1 - one tablet contains:                                      ______________________________________                                        active substance         20 mg                                                microcrystalline cellulose                                                                            150 mg                                                lactose                  20 mg                                                amide                    10 mg                                                magnesium stearate       5 mg                                                 ______________________________________                                    

The active substance is chosen from the group formed by the gangliosidederivatives described in any one of Examples 9, 13, 19 and 26.

    ______________________________________                                        Preparation No. 2 - one pill contains:                                        ______________________________________                                        active substance         30 mg                                                carboxymethyl cellulose 150 mg                                                amide                    15 mg                                                shellac                  10 mg                                                saccharose               35 mg                                                coloring                 0.5 mg                                               ______________________________________                                    

The active substance is chosen from the group formed by the gangliosidederivatives described in any one of Examples 9, 14 and 28.

    ______________________________________                                        Preparation No. 3 one gelatinous capsule contains:                            ______________________________________                                        active substance         40 mg                                                lactose                 100 mg                                                gastroresistant coating  5 mg                                                 ______________________________________                                    

The active substance is chosen from the group formed by the gangliosidederivatives described in any one of Examples 15, 24 and 24.

    ______________________________________                                        Preparation No. 4 - one soft gelatin capsule contains:                        ______________________________________                                        active substance        50 mg                                                 vegetable oil          200 mg                                                 beeswax                 20 mg                                                 gelatin                150 mg                                                 glycerol                50 mg                                                 coloring                3 mg                                                  ______________________________________                                    

The active substance is chosen from the group formed by the gangliosidederivatives described in any one of Examples 15, 24 and 34.

The following is claimed:
 1. A pharmaceutical composition comprising atherapeutically effective amount ofN-(2-thiopheneacetyl)-N'-acetyl-N,N'-di-lyso GM₁ as the activeingredient, together with a pharmaceutically acceptable excipient.
 2. AnN,N'-diacyl-N,N'-di-lysoganglioside which isN-(2-thiopheneacetyl)-N'-acetyl-N,N'-di-lyso GM₁.